Anti-cd40 antibodies and methods of use

ABSTRACT

The present invention provides high affinity anti-CD40 monoclonal antibodies and related compositions, which may be used in any of a variety of therapeutic methods for the treatment of cancer and other diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/594,367, filed May 12, 2017, now allowed, which is divisional of U.S.patent application Ser. No. 14/067,770, filed on Oct. 30, 2013, issuedas U.S. Pat. No. 9,676,861, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/720,289 filed, on Oct. 30,2012, where these applications are herein incorporated by reference intheir entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is APEX_016_03US_ST25.txt. The text file is 93 KB,was created on Mar. 12, 2018 and is being submitted electronically viaEFS-Web.

BACKGROUND Technical Field

The present invention relates generally to anti-CD40 antibodies,compositions and methods of using same. Such antibodies are useful, forexample, in methods for treating a variety of oncological diseases.

Description of the Related Art

The majority of leukemias and lymphomas originate from malignanttransformation of B-lineage cells. The expression of cell surfaceB-lineage-restricted antigens such as CD20 makes it an attractive targetfor antibody therapy. Antibody therapeutics have dramatically changedthe management of patients with non-Hodgkin lymphoma (NHL) and chroniclymphocytic leukemia (CLL). Since the approval of rituximab, theantibody alone or in combination with chemotherapy has remarkablyimproved response rates, long-term outcomes, and quality of life (ChinnP, Braslawsky G, White C, et al. Antibody therapy of non-Hodgkin'sB-cell lymphoma. Cancer Immunol Immunother 2003; 52:257-280; RastetterW, Molina A, White C A. Rituximab: Expanding role in therapy forlymphomas and autoimmune diseases. Annu Rev Med 2004; 55:477-503).However, a substantial number of patients exhibit either primary oracquired resistance to rituximab, suggesting that current approachestargeting CD20 have limitations in clinical outcomes, and there is aneed for improvement by developing novel immunotherapeutics for B celllymphoma and leukemia with distinct mechanisms of action (Stolz C,Schuler M. Molecular mechanisms of resistance to Rituximab andpharmacologic strategies for its circumvention. Leukemia and lymphoma.2009; 50(6):873-885; Bello C, Sotomayor E M. Monoclonal antibodies forB-cell lymphomas: Rituximab and beyond. Hematology Am Soc Hematol EducProgram 2007; 233-242; Dupire S, Coiffier B. Targeted treatment and newagents in diffuse large B cell lymphoma. Int J Hematol 2010; June 18(online)), such as the anti-CD40 mAb, APX005.

The Role of CD40 in the Regulation of Immune Responses

Full activation of T cells requires two distinct but synergisticsignals. The first signal, delivered through the T-cell antigenreceptor, is provided by antigen and MHC complex on APCs and isresponsible for the specificity of the immune response. The secondary,or costimulatory signal is through the interaction of CD28 with B7-1(CD80)/B7-2 (CD86), and CD40 with CD40L, which are required to mount afull scale T cell response. In the absence of costimulatory signals, Tcells may undergo unresponsiveness (anergy) or programmed cell death(apoptosis) upon antigen stimulation.

CD40, a member of the TNF receptor (TNFR) superfamily, is expressedprimarily on B cells and other antigen-presenting cells (APCs) such asdendritic cells and macrophages. CD40 ligand (CD40L) is expressedprimarily by activated T cells.

CD40 and CD40L interaction serves as a costimulatory signal for T cellactivation. CD40-CD40L engagement on resting B cells inducesproliferation, immunoglobulin class switching, antibody secretion, andalso has a role in the development of germinal centers and the survivalof memory B cells, all of which are essential to humoral immuneresponses (Kehry M R. J Immunol 1996; 156: 2345-2348). Binding of CD40Lto CD40 on dendritic cells induces DC maturation as manifested byincreasing expression of co-stimulatory molecules such as B7 family(CD80, CD86) and production of proinflammatory cytokines such asinterleukin 12. These lead to potent T cell responses (Stout, R. D., J.Suttles. 1996. Immunol. Today 17:487-492; Brendan O'Sullivan, RanjenyThomas. Critical Reviews in Immunology 2003; 23: 83-107; Cella, M., D.Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. J.Exp. Med. 1996; 184:747-452).

CD40 signal transduction activates multiple pathways including NF-KappaB(Nuclear Factor-KappaB), MAPK (Mitogen-Activated Protein Kinase) andSTAT3 (Signal Transducers and Activators of Transcription-3) (Pype S, etal. J Biol Chem. 2000 Jun. 16; 275(24):18586-93) that regulate geneexpression through activation of Activating Proteins, c-Jun, ATF2(Activating Transcription Factor-2) and Rel transcription factors(Dadgostar H, et al. Proc Natl Acad Sci USA. 2002 Feb. 5;99(3):1497-502). The TNFR-receptor associated factor adaptor proteins(e.g., TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6) interact with thisreceptor and serve as mediators of the signal transduction. Depending onthe particular cell type, CD40 engagement results in a particular geneexpression pattern. Genes activated in response to CD40 signallinginclude numerous cytokines and chemokines (IL-1, IL-6, IL-8, IL-10,IL-12, TNF-Alpha, and Macrophage Inflammatory Protein-1Alpha(MIP1Alpha). In certain cell types, activation of CD40 may result inproduction of cytotoxic radicals (Dadgostar et al., Supra), COX2(Cyclooxygenase-2), and production of NO (Nitric Oxide).

The Role of CD40 in Tumors

CD40 is not only expressed by normal immune cells but also by manymalignant cells. In particular, CD40 is over-expressed in B-lineageNHLs, chronic lymphocytic leukemias (CLLs), hairy cell leukemias (HCLs),Hodgkin's disease (Uckun F M, Gajl-Peczalska K, Myers D E, et al. Blood1990; 76:2449-2456; O'Grady J T, Stewart S, Lowrey J, et al. Am J Pathol1994; 144: 21-26), multiple myeloma (Pellat-Deceunynck C, Bataille R,Robillard N, Harousseau J L, Rapp M J, Juge-Morineau N, Wijdenes J,Amiot M. Blood. 1994; 84(8):2597-603), as well as in carcinomas of thebladder, kidney, ovary, cervix, breast, lung, nasopharynx, and malignantmelanoma (Young L S, Eliopoulos A G, Gallagher N J, et al. Immunol Today1998; 19:502-6; Ziebold J L, Hixon J, Boyd A, et al. Arch Immunol TherExp (Warsz) 2000; 48: 225-33; Gladue R, Cole S, Donovan C, et al. J ClinOncol 2006; 24 (18S):103s).

Ligation of CD40 on the surface of tumor cells, which in many cases,mediates a direct cytotoxic effect, results in tumor regression throughapoptosis and necrosis (Grewal I S, Flavell R A. Annu Rev Immunol 1998;16:111-35; van Kooten C, Banchereau J. J Leukoc Biol 2000; 67(1):2-17).Although the exact functions of CD40 in tumor cells are unclear (Tong AW, Stone M J. Cancer Gene Ther. 2003 10(1):1-13), engagement of CD40 invitro inhibits the growth of solid tumor cells and high-grade B celllymphoma cells (Magi Khalil and Robert H. Vonderheide. Update CancerTher 2007; 2(2): 61-65; Young L S, Eliopoulos A G, Gallagher N J, DawsonC W. Immunol Today 1998; 19(11):502-6; Funakoshi S, Longo D L, BeckwithM, et al. Blood 1994; 83(10):2787-94; Hess S, Engelmann H. J Exp Med1996; 183(1):159-67; Eliopoulos A G, Dawson C W, Mosialos G, et al.Oncogene 1996; 13(10):2243-54; von Leoprechting A, van der Bruggen P,Pahl H L, Aruffo A, Simon J C. Cancer Res 1999; 59(6):1287-94). Theseeffects contrast with proliferation induced after engagement of CD40 onnon-neoplastic B cells and dendritic cells.

In addition to direct tumor inhibition, activation of CD40 signalingrescues the function of antigen-presenting cells in tumor-bearing hostsand triggers or restores active immune responses againsttumor-associated antigens. CD40 agonists have been reported to overcomeT-cell tolerance in tumor-bearing mice, evoke effective cytotoxic T-cellresponses against tumor-associated antigens, and enhance the efficacy ofantitumor vaccines (Eliopoulos A G, Davies C, Knox P G, et al. Mol CellBiol 2000; 20(15): 5503-15; Tong A W, Papayoti M H, Netto G, et al. ClinCancer Res 2001; 7(3):691-703).

CD40 as Molecular Target

CD40 is overexpressed on a wide range of malignant cells. The roles ofCD40 in tumor inhibition and stimulation of the immune system make CD40an attractive target for an antibody-based immunotherapy (van Mierlo GJ, den Boer A T, Medema J P, et al. Proc Natl Acad Sci USA. 2002; 99(8):5561-5566; French R R, Chan H T, Tutt A L, Glennie M J. Nat Med. 1999;5(5):548-553). Anti-CD40 antibodies may act against cancer cells viamultiple mechanisms: (i) antibody effector function such as ADCC, (ii) adirect cytotoxic effect on the tumor cells, and (iii) activation ofanti-tumor immune responses.

Anti-CD40 Therapeutic Antibodies in Development

Several anti-CD40 antibodies have been reported to have potential asanti-tumor therapeutics. CP-870,893 is a fully human IgG2 CD40 agonistantibody developed by Pfizer. It binds CD40 with a K_(D) of 3.48×10⁻¹⁰M, but does not block binding of CD40L (see e.g., U.S. Pat. No.7,338,660). CP-870893 has not shown ADCC effects; possibly due to itsIgG2 isotype. Thus, this antibody acts as a CD40 agonist (i.e., does notaffect CD40L binding), induces proapoptotic signaling, and activates DCsand immune surveillance. However, this antibody does not mediate ADCC.

HCD122 is a fully human IgG1 CD40 antagonist antibody developed byNovartis. It binds to CD40 with a K_(D) of 5.1×10⁻¹⁰ M, blocks CD40binding to CD40L, inhibits CD40-ligand induced signaling and biologicaleffects on B cells and certain primary CLL and MM cells (Tai Y T, et al.Cancer Res. 2005 Jul. 1; 65(13):5898-906; Luqman M, Klabunde S, et al:Blood 112:711-720, 2008). The major mechanism of action for itsanti-tumor effect in vivo is ADCC (Long L, et al. 2005 IMF OralPresentation and Abstract No. 3; Blood 2004, 104(11, Part 1): Abst3281). Due to its antagonist feature, this antibody may not directlyinduce CD40-mediated anti-tumor immune response.

SGN-40 is a humanized IgG1 antibody developed by Seattle Genetics frommouse antibody clone S2C6, which was generated using a human bladdercarcinoma cell line as the immunogen. It binds to CD40 with a K_(D) of1.0×10⁻⁹ M and works through enhancing the interaction between CD40 andCD40L, thus exhibiting a partial agonist effect (Francisco J A, et al.,Cancer Res, 60: 3225-31, 2000). SGN-40 delivers proliferation inhibitoryand apoptosis signals to a panel of B lymphoma lines originated fromhigh-grade non-Hodgkin's lymphoma and MM cells (Tai Y T, Catley L P,Mitsiades C S, et al. Cancer Res 2004; 64(8):2846-2852). In vitro and invivo studies suggest that both apoptotic signaling and antibody effectorfunction via ADCC contribute to antitumor activity of SGN-40 (Law C L,Gordon K A, Collier J, et al: Cancer Res 2005; 65:8331-8338). A Recentstudy suggested that the anti-tumour activity of SGN-40 significantlydepends on Fc interactions with the effector cells and that macrophagesare the major effectors contributing to its therapeutic activities(Oflazoglu E, et al. Br J Cancer. 2009 Jan. 13; 100(1):113-7. Epub 2008Dec. 9). Since SGN-40 is a partial agonist and requires CD40L expressedon T cells, SGN-40 may have limited ability to fully boost theanti-tumor immune response.

Accordingly, there remains a need in the art for novelimmunotherapeutics that target CD40 and that act as agonist for thistarget, activate dendritic cells and immune surveillance and whichactivate ADCC, thereby providing improved anti-cancer properties.

BRIEF SUMMARY

One aspect of the present disclosure provides an isolated antibody, oran antigen-binding fragment thereof, that binds to human CD40,comprising (i) a heavy chain variable region comprising the VHCDR1region set forth in SEQ ID NO:3, the VHCDR2 region set forth in SEQ IDNO:4, and the VHCDR3 region set forth SEQ ID NO:5; and (ii) a lightchain variable region comprising the VLCDR1 region set forth in SEQ IDNO:6, the VLCDR2 region set forth in SEQ ID NO:7, and the VLCDR3 regionset forth in SEQ ID NO: 8; or a variant of said antibody, or anantigen-binding fragment thereof, comprising heavy and light chainvariable regions identical to the heavy and light chain variable regionsof (i) and (ii) except for up to 8 amino acid substitutions in said CDRregions. In one embodiment of the antibodies disclosed herein, the heavychain variable region comprises the amino acid sequence set forth in SEQID NO:1. In a further embodiment, the light chain variable regioncomprises the amino acid sequence set forth in SEQ ID NO:2.

Another aspect of the present disclosure provides an isolated antibody,or an antigen-binding fragment thereof, that binds to human CD40,comprising a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO:1. In one embodiment of this aspect, theisolated antibody, or antigen-binding fragment thereof comprises a lightchain variable region which comprises an amino acid sequence having atleast 90% identity to the amino acid sequence set forth in SEQ ID NO:2.In a further embodiment of this aspect, the isolated antibody, or anantigen-binding fragment thereof comprises a light chain variable regionwhich comprises the amino acid sequence set forth in SEQ ID NO:2.

Yet a further aspect of the present disclosure provides an isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprising a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:2. In one embodiment of this aspect, theisolated antibody, or antigen binding fragment thereof comprises a heavychain variable region which comprises an amino acid sequence having atleast 90% identity to the amino acid sequence set forth in SEQ ID NO:1.

In certain embodiments, the isolated antibodies as disclosed herein arehumanized. Illustrative humanized antibody variable regions are setforth in the VH region amino acid sequence of SEQ ID NO:9 and the VLregion amino acid sequence of SEQ ID NO:10.

In one embodiment, an isolated antibody disclosed herein may be singlechain antibody, a ScFv, a univalent antibody lacking a hinge region, aminibody, a Fab, a Fab′ fragment, or a F(ab′)₂ fragment. In certainembodiments, the antibodies herein are whole antibodies.

In another embodiment, the isolated antibodies as described hereincomprise a human IgG constant domain, such as, but not limited to anIgG1 CH1 domain or an IgG1 Fc region.

A further embodiment of the disclosure provides an isolated antibody, oran antigen-binding fragment thereof, that competes with the anti-CD40antibodies described herein for binding to human CD40.

In one aspect of this disclosure, the isolated antibody, orantigen-binding fragment thereof, that binds CD40, binds with a KD of0.96 nM or lower. In a further embodiment, the isolated antibody, orantigen-binding fragment thereof, that binds CD40, binds with a Kd ofbetween 1.1 nM and 0.90 nM. In a further embodiment, the isolatedantibody, or antigen-binding fragment thereof, that binds CD40, bindswith a Kd of about 1.2, 1.1, 1.0, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94,0.93, 0.92, 0.91, 0.90, 0.85, or about 0.80 nM. In another embodiment,the antibody binds CD40 with a Kd of about 2.5, 2.4, 2.3, 2.2, 2.1, 2.0,1.9, 1.8, 1.7, 1.6, 1.5, 1.4, or 1.3 nM.

In a further aspect, the invention provides an isolated antibody, orantigen-binding fragment thereof as described herein, wherein theisolated antibody, or antigen-binding fragment thereof: blocks bindingof CD40 to CD40L; is a CD40 agonist; activates antigen presenting cells;stimulates cytokine release from antigen presenting cells; induces tumorcell apoptosis; inhibits tumor cell proliferation; kills tumor cells viainduction of effector functions selected from the group consisting ofantibody dependent cellular cytotoxicity, complement dependentcytotoxicty, and antibody dependent cellular phagocytosis; stimulatesanti-tumor T cell responses; reduces established tumors; inhibitsrituximab-resistant tumors; or a combination of any one or more of theaforementioned.

Another aspect of the present invention provides an isolated antibody,or an antigen binding fragment thereof, that binds to CD40, comprising:(i) a heavy chain variable region comprising the VH CDR1, the VHCDR2,and VHCDR3 of any one of the VH regions shown in FIG. 16; and (ii) alight chain variable region comprising the VLCDR1, the VLCDR2, and theVLCDR3 region of the corresponding VL region of any one of the VLregions shown in FIG. 16; or a variant of said antibody, or an antigenbinding fragment thereof, comprising heavy and light chain variableregions identical to the heavy and light chain variable regions of (i)and (ii) except for up to 8 amino acid substitutions in said CDRregions.

Yet another aspect of the present invention provides an isolatedantibody, or an antigen binding fragment thereof that binds to CD40,comprising a heavy chain variable region comprising any one of the VHregions shown in FIG. 16. In one embodiment such an antibody furthercomprises a light chain variable region comprising an amino acidsequence having at least 90% identity to the corresponding VL region asshown in FIG. 16. In another embodiment, such an antibody or antigenbinding fragment thereof further comprises the corresponding light chainvariable region as shown in FIG. 16.

Yet another aspect of the present invention provides an isolatedantibody, or an antigen binding fragment thereof that binds to CD40,comprising a light chain variable region comprising any one of the VLregions shown in FIG. 16. In one embodiment such an antibody furthercomprises a heavy chain variable region comprising an amino acidsequence having at least 90% identity to the corresponding VH region asshown in FIG. 16. In another embodiment, such an antibody or antigenbinding fragment thereof further comprises the corresponding heavy chainvariable region as shown in FIG. 16.

The present disclosure also provides isolated polynucleotides encodingthe isolated antibodies, or antigen-binding fragments thereof asdisclosed herein.

The present disclosure also provides compositions comprising aphysiologically acceptable carrier and a therapeutically effectiveamount of an anti-CD40 antibody or antigen-binding fragment thereof asdescribed herein.

Another aspect of the present disclosure provides a method for treatinga patient having a cancer, comprising administering to the patient acomposition comprising a physiologically acceptable carrier and atherapeutically effective amount of an anti-CD40 antibody orantigen-binding fragment thereof as described herein, thereby treatingthe cancer. In certain embodiments, the cancer is associated withaberrant CD40 expression. In further embodiments, the cancer is selectedfrom the group consisting of non-Hodgkin's lymphomas, Hodgkin'slymphoma, chronic lymphocytic leukemias, hairy cell leukemias, acutelymphoblastic leukemias, multiple myeloma, carcinomas of the pancreas,colon, gastric intestine, prostate, bladder, kidney, ovary, cervix,breast, lung, nasopharynx, malignant melanoma and rituximab resistantNHL and leukemias.

Another aspect of the present disclosure provides a method for treatinga patient having cancer and/or autoimmune disease, and/or inflammatorydisease, comprising administering to the patient a compositioncomprising a physiologically acceptable carrier and a therapeuticallyeffective amount of an anti-CD40 antibody or antigen-binding fragmentthereof as described herein, thereby treating the patient havingautoimmune and inflammatory dieseases.

Another aspect of the present disclosure provides a method forameliorating the symptoms in a patient having cancer, and/or autoimmunedisease and/or inflammatory disease, comprising administering to thepatient a composition comprising a physiologically acceptable carrierand a therapeutically effective amount of an anti-CD40 antibody orantigen-binding fragment thereof as described herein, therebyameliorating the symptoms in the patient having cancer, and/orautoimmune and/or inflammatory diseases.

Another aspect of the present disclosure provides isolated antibody, oran antigen-binding fragment thereof, that binds to human CD40,comprising a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO:11. In one embodiment, the isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprises a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO:11 and comprises a light chain variableregion which comprises an amino acid sequence having at least 90%identity to the amino acid sequence set forth in SEQ ID NO:22 or a lightchain comprising the amino acid sequence set forth in SEQ ID NO:22. Incertain embodiments, an isolated antibody described herein comprises thelight chain as set forth in SEQ ID NO:22 and comprises a heavy chainvariable region which comprises an amino acid sequence having at least90% identity to the amino acid sequence set forth in SEQ ID NO:11.

A further aspect of the present disclosure provides an isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprising a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO:13. In one embodiment, the antibodycomprises a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO:13 and a light chain variable regionwhich comprises an amino acid sequence having at least 90% identity tothe amino acid sequence set forth in SEQ ID NO:24. In one embodiment,the light chain comprises the amino acid sequence set forth in SEQ IDNO:24.

A further aspect of the present disclosure provides an isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprising a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:24. In one embodiment, the antibodycomprises a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:24 and a heavy chain variable regionwhich comprises an amino acid sequence having at least 90% identity tothe amino acid sequence set forth in SEQ ID NO:13.

In certain aspects, the isolated antibody, or antigen-binding fragmentthereof, that binds CD40 comprises a heavy chain variable region whichcomprises the amino acid sequence set forth in SEQ ID NO:17. In oneembodiment, the isolated antibody that binds CD40 comprises a heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO:17 and a light chain variable region which comprises an aminoacid sequence having at least 90% identity to the amino acid sequenceset forth in SEQ ID NO:28. In one embodiment, the light chain variableregion comprises the amino acid sequence set forth in SEQ ID NO:28.

Another aspect of the disclosure provides an isolated antibody, or anantigen-binding fragment thereof, that binds to human CD40, comprising alight chain variable region comprising the amino acid sequence set forthin SEQ ID NO:28. In one embodiment, the isolated antibody, or anantigen-binding fragment thereof, that binds to human CD40, comprises alight chain variable region comprising the amino acid sequence set forthin SEQ ID NO:28 and a heavy chain variable region which comprises anamino acid sequence having at least 90% identity to the amino acidsequence set forth in SEQ ID NO:17.

Another aspect of the disclosure provides an isolated antibody, or anantigen-binding fragment thereof, that binds to human CD40, comprising aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO:19. In one embodiment, the isolated antibody, or anantigen-binding fragment thereof, that binds to human CD40, comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO:19 and a light chain variable region which comprises anamino acid sequence having at least 90% identity to the amino acidsequence set forth in SEQ ID NO:30. In one particular embodiment, thelight chain variable region comprises the amino acid sequence set forthin SEQ ID NO:30.

Yet a further aspect of the present disclosure provides an isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprising a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:30. In one embodiment, the isolatedantibody, or an antigen-binding fragment thereof, that binds to humanCD40, comprises a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:30 and a heavy chain variable regionwhich comprises an amino acid sequence having at least 90% identity tothe amino acid sequence set forth in SEQ ID NO:19.

Another aspect of the present disclosure provides an isolated antibody,or an antigen-binding fragment thereof, that binds to human CD40,comprising a heavy chain variable region that comprises heavy chainvariable region CDRs and a light chain variable region that comprisescorresponding light chain variable region CDRs, wherein the CDRs are asshown in FIG. 16.

Another aspect of the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to a CD40 epitope setforth in any one or more of SEQ ID NOs: 196, 197, 199 and 202. In oneparticular embodiment, the isolated antibody, or antigen bindingfragment thereof binds to the CD40 epitope set forth in SEQ ID NO: 202.In another embodiment, an isolated antibody, or antigen-binding fragmentthereof, that binds to the CD40 epitopes set forth in SEQ ID NOs: 196,197, 199 or 202, does not comprise the CDRs set forth in SEQ ID NOs:3-8.In one embodiment, the isolated antibody that binds to a CD40 epitopeset forth in any one or more of SEQ ID NOs: 196, 197, 199 and 202,comprises (i) a heavy chain variable region comprising the VHCDR1 regionset forth in SEQ ID NO:3, the VHCDR2 region set forth in SEQ ID NO:4,and the VHCDR3 region set forth SEQ ID NO:5; and (ii) a light chainvariable region comprising the VLCDR1 region set forth in SEQ ID NO:6,the VLCDR2 region set forth in SEQ ID NO:7, and the VLCDR3 region setforth in SEQ ID NO: 8; or a variant of said antibody, or anantigen-binding fragment thereof, comprising heavy and light chainvariable regions identical to the heavy and light chain variable regionsof (i) and (ii) except for up to 8 amino acid substitutions in said CDRregions; said isolated antibody, or an antigen-binding fragment thereof,further comprising an Fc region modified such that the isolatedantibody, or an antigen binding fragment thereof, has increased bindingaffinity to FcγRIIB, increased ADCC, or increased anti-CD40 agonistactivity, or a combination thereof, as compared to said isolatedantibody, or an antigen binding fragment thereof, comprising anunmodified version of said Fc region. In this regard, the Fc region maycomprise a S267E mutation.

The present disclosure also provides isolated polynucleotides encodingthe isolated antibodies, or antigen-binding fragments thereof, that bindto a CD40 epitope set forth in any one or more of SEQ ID NOs: 196, 197,199 and 202; expection vectors comprising the isolated polynucleotide,and isolated host cells comprising such vector.

The present disclosure also provides compositions comprising aphysiologically except a carrier and a therapeutically effective amountof the isolated antibodies, or antigen binding fragment thereof, thatbind to a CD40 epitope set forth in any one or more of SEQ ID NOs: 196,197, 199 and 202

The present invention further provides methods for treating orameliorating cancer in a patient comprising administering to the patientthe compositions comprising the antibodies that bind to a CD40 epitopeset forth in any one or more of SEQ ID NOs: 196, 197, 199 and 202, asdescribed herein. In this regard, the cancer may be non-Hodgkin'slymphomas, Hodgkin's lymphoma, chronic lymphocytic leukemias, hairy cellleukemias, acute lymphoblastic leukemias, multiple myeloma, carcinomasof the bladder, kidney ovary, cervix, breast, lung, nasopharynx,malignant melanoma or rituximab resistant NHL or leukemias.

The present invention also provides methods for ameliorating symptoms ofautoimmune disease or inflammatory disease in a patient by administeringto the patient the compositions comprising the antibodies that bind to aCD40 epitope set forth in any one or more of SEQ ID NOs: 196, 197, 199and 202.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of the VH region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:2 is the amino acid sequence of the VL region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:3 is the amino acid sequence of the VHCDR1 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:4 is the amino acid sequence of the VHCDR2 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:5 is the amino acid sequence of the VHCDR3 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:6 is the amino acid sequence of the VLCDR1 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:7 is the amino acid sequence of the VLCDR2 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:8 is the amino acid sequence of the VLCDR3 region of the R-8rabbit anti-CD40 antibody.

SEQ ID NO:9 is the amino acid sequence of the VH region of APX005, thehumanized version of the R-8 rabbit anti-CD40 antibody, without a signalpeptide.

SEQ ID NO:10 is the amino acid sequence of the VL region of APX005, thehumanized version of the R-8 rabbit anti-CD40 antibody, without a signalpeptide.

SEQ ID NOs:11-21 and 33-44 are heavy chain amino acid sequences ofrabbit anti-CD40 antibody candidates that showed functional activity(see FIG. 16).

SEQ ID NOs:22-32 and 45-56 are light chain amino acid sequences ofrabbit anti-CD40 antibody candidates that showed functional activity(see FIG. 16).

SEQ ID Nos:57-79 are the VHCDR1 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID Nos:80-102 are the VHCDR2 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID Nos:103-125 are the VHCDR3 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID Nos:126-148 are the VLCDR1 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID Nos:149-171 are the VLCDR2 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID Nos:172-194 are the VLCDR3 amino acid sequences for the anti-CD40antibodies shown in FIG. 16.

SEQ ID NO:195 is the amino acid sequence of the human IgG1 heavy chainconstant region comprising an Fc region with a S267E substitution.

SEQ ID NO: 196 is amino acids 92-107 of human CD40, identified as anepitope bound by the APX005 antibody.

SEQ ID NO: 197 is amino acids 125-144 of human CD40, identified as anepitope bound by the APX005 antibody.

SEQ ID NO:198 is the amino acid sequence of human CD40.

SEQ ID NO: 199 is the amino acid sequence of a CD40 CLIPS peptide, fromresidue 84-102 of CD40.

SEQ ID NO:200 is the amino acid sequence of residues 84-102 of CD40withserine substitutions for the cysteine residues at position 91 and96.

SEQ ID NO:201 is the amino acid residues 122-125 of human CD40.

SEQ ID NO:202 is the amino acid residues 92-102 of human CD40 bound bythe APX005 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D show the results of screening agonist antibodies by measuringDC maturation and T cell activation as described in Example 1. 1A: CD83expression; 1B: CD80 expression; 1C: CD86 expression; 1 D: T cellproliferation in a mixed lymphocyte reaction.

FIG. 2 is a graph showing the comparison of lead candidates in theinhibition of Ramos cell proliferation.

FIG. 3 is a bar graph showing the results of an ADCC assay. Effector(human PBMC):target cell (Ramos cell) ratio of 40:1.

FIG. 4A and FIG. 4B are graphs showing the results of in vivo screeningof anti-tumor activity of anti-CD40 candidates.

FIG. 5 is a graph showing the results of an ELISA assay demonstratingthat APX005 selectively binds to CD40 but not to other TNFR familymembers.

FIG. 6 is a graph showing the results of an ELISA assay demonstratingthat APX005 blocks the binding of CD40L to CD40.

FIG. 7 is a graph showing that APX005 is not Internalized upon Bindingto CD40 positive cells.

FIG. 8A and FIG. 8B are graphs showing APX005-mediated ADCC of CD40positive Ramos (FIG. 8A) and Daudi (FIG. 8B) tumor cells.

FIG. 9A and FIG. 9B are graphs showing in vitro inhibition of Ramostumor cell proliferation by APX005. Panel A: without Fc crosslinking;Panel B: with Fc crosslinking.

FIG. 10 is a bar graph showing induction of DC activation by APX005.

FIGS. 11A and 11B show that APX005 binds to human and monkey CD40 butnot mouse CD40.

FIG. 12A is a graph showing APX005 inhibition of tumor growth in a Ramosmodel. FIG. 12B is a bar graph showing levels of serum human IgG in miceat day 34, two days after the last dosing.

FIGS. 13A and 13B are graphs showing inhibition of Rituximab pre-treatedand resistant tumors in a mouse model.

FIG. 14 is a graph showing APX005 inhibition of tumor growth in the Rajimouse model.

FIG. 15 is a graph showing potent anti-tumor activity of APX005 againsthuman multiple myeloma in the IM-9 xenograft model.

FIG. 16A-16L is a sequence alignment of rabbit anti-CD40 heavy (FIGS.16A-16F) and light chain (FIGS. 16G-16L) antibody sequences. Heavy andlight chain CDRs1-3 are underlined. SEQ ID NOs are as follows: Heavychain: R-3 and R-6: SEQ ID NOs:11, 12; R-8: SEQ ID NO:1; R-9, -16, -18,-24, -33, -36, 19-21, -45, -59: SEQ ID NOs:13-21, respectively; R-2,R-5, R-7, R-10, R-12, R-20, R-26, R-30, R-35, 19-35, 19-41, 19-57: SEQID Nos:33-44, respectively. Light chain: R-3 and R-6: SEQ ID NOs:22 and23; R-8: SEQ ID NO:2; R-9, -16, -18, -24, -33, -36, 19-21, -45, -59: SEQID NOs:24-32, respectively; R-2, R-5, R-7, R-10, R-12, R-20, R-26, R-30,R-35, 19-35, 19-41, 19-57: SEQ ID Nos:45-56, respectively. The aminoacid sequences include the VH and VL signal peptide. The R-8 VHCDR andVLCDR amino acid sequences are set forth in SEQ ID Nos:3-8. The VHCDRamino acid sequences and the VLCDR amino acid sequences for theremaining antibodies are set forth in SEQ ID Nos:57-125 and SEQ IDNos:126-194, respectively.

FIGS. 17A and 17B show inhibition of tumor growth in the ramos model byAPX005 as compared with SGN-40 and Rituximab.

FIGS. 18A and 18B show inhibition by APX005 of tumor growth inrituximab-resistant human Namalwa lymphoma xenograft model.

FIG. 19 shows a comparison of APX005 and APX005S267E binding to CD40.

FIG. 20 shows binding affinity of APX005 and APX005 S267E mutant ascompared with other anti-CD40 antibodies.

FIG. 21 shows CD40 agonist activity of APX005 S267E mutant. The EC50(ng/ml) for this experiment was as follows: APX005: 24.35; APX005 S267E:4.15; SGN-40: 1264.00; CP870893: 37.00; 19-21: 74.65.

FIG. 22 shows ADCC activity of the APX005 S267E mutant.

FIG. 23 shows the visualization of the APX005 epitopes on the cartoonrendering (FIG. 23A) and surface rendering (FIG. 23B) of the CD40structure 3QD6. The epitope area ₉₂TSEACESCVLHRSCSP₁₀₇ (SEQ ID NO: 196)is mapped onto the crystal structure of CD40 and is outlined in bold.Residues 125-144, which were also found to be bound by the APX005antibody, are not resolved in structure 3QD6, but based on the availabledata on cysteine bonds, are expected to be in close proximity toresidues 92-107. Therefore, for reference and visualization purposes,residues 122-125 (SEQ ID NO:201) are shown.

DETAILED DESCRIPTION

The present disclosure relates to antibodies and antigen-bindingfragments thereof the specifically bind to CD40 in particular antibodieshaving specific epitopic specificity and functional properties. Oneembodiment of the invention encompasses specific humanized antibodiesand fragments thereof capable of binding to CD40 and functions as a CD40agonist by inducing/enhancing CD40-mediated downstream cell signalingand biological effects. In more specific embodiments of the invention,the antibodies described herein specifically bind to CD40 with very highaffinity, such as an affinity of at least between 980 and 950 picomolar,at least between 970 and 950 picomolar, and in certain embodiments withan affinity of 960 picomolar. The antibodies described herein, amongother attributes, induce CD40 signalling in tumor cells, activatedendritic cells and immune surveillance, activate antibody dependentcellular cytotoxicity (ADCC) against tumor cells, block binding of CD40to CD40L; have CD40 agonistic activity; activate antigen presentingcells; stimulate cytokine release from antigen presenting cells; inducetumor cell apoptosis; Inhibit tumor cell proliferation; kill tumor cellsvia induction of effector functions including but not limited to ADCC,CDC and ADCP; stimulate anti-tumor T cell responses; reduce establishedtumors; and inhibit rituximab-resistant tumors. The antibodies describedherein may have or induce a combination of any one or more of theseattributes or activities.

Embodiments of the invention pertain to the use of anti-CD40 antibodiesor antigen-binding fragments thereof for the diagnosis, assessment andtreatment of diseases and disorders associated with CD40 or aberrantexpression thereof. The subject antibodies are used in the treatment orprevention of cancers including, but not limited to, non-Hodgkin'slymphomas, Hodgkin's lymphoma, chronic lymphocytic leukemias, hairy cellleukemias, acute lymphoblastic leukemias, multiple myeloma, carcinomasof the bladder, kidney ovary, cervix, breast, lung, nasopharynx,malignant melanoma and rituximab resistant NHL and leukemias, autoimmunediseases and inflammatory diseases among other diseases.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in MolecularBiology or Current Protocols in Immunology, John Wiley & Sons, New York,N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology,3^(rd) ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning:A Laboratory Manual (3rd Edition, 2001); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984) and other like references.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

Embodiments of the present invention relate to antibodies that bind toCD40. In particular, the antibodies described herein specifically bindto CD40 with unexpectedly high affinity, enhance CD40 signallingactivity, activate the immune system, activate ADCC and have therapeuticutility for the treatment of diseases associated with aberrantexpression CD40.

Sequences of illustrative antibodies, or antigen-binding fragments, orcomplementarity determining regions (CDRs) thereof, are set forth in SEQID NOs:1-194.

As is well known in the art, an antibody is an immunoglobulin moleculecapable of specific binding to a target, such as a carbohydrate,polynucleotide, lipid, polypeptide, etc., through at least one epitoperecognition site, located in the variable region of the immunoglobulinmolecule. As used herein, the term encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)2, Fv), single chain (ScFv), synthetic variantsthereof, naturally occurring variants, fusion proteins comprising anantibody portion with an antigen-binding fragment of the requiredspecificity, humanized antibodies, chimeric antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity. “Diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; P. Holliger et al.,Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993) are also a particularform of antibody contemplated herein. Minibodies comprising a scFvjoined to a CH3 domain are also included herein (S. Hu et al., CancerRes., 56, 3055-3061, 1996). See e.g., Ward, E. S. et al., Nature 341,544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al.,PNAS USA, 85, 5879-5883, 1988); PCT/US92/09965; WO94/13804; P. Holligeret al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993; Y. Reiter et al.,Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56,3055-3061, 1996.

The term “antigen-binding fragment” as used herein refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chains that binds to the antigen of interest, inparticular to CD40. In this regard, an antigen-binding fragment of theherein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs ofa VH and VL sequence set forth herein from antibodies that bind CD40. Anantigen-binding fragment of the CD40-specific antibodies describedherein is capable of binding to CD40. In certain embodiments, anantigen-binding fragment or an antibody comprising an antigen-bindingfragment, prevents or inhibits CD40L binding to the CD40. In certainembodiments, the antigen-binding fragment binds specifically to and/orenhances or modulates the biological activity of human CD40. Suchbiological activity includes, but is not limited to, cell signalling,activation of dendritic cells,

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. An epitope is a region of an antigen that is bound by anantibody. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl, and may in certainembodiments have specific three-dimensional structural characteristics,and/or specific charge characteristics. In certain embodiments, anantibody is said to specifically bind an antigen when it preferentiallyrecognizes its target antigen in a complex mixture of proteins and/ormacromolecules. An antibody is said to specifically bind an antigen whenthe equilibrium dissociation constant is <10⁻⁷ or 10⁻⁸ M. In someembodiments, the equilibrium dissociation constant may be <10⁻⁹ M or<10⁻¹⁰ M.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures-regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof, now available on the Internet(immuno.bme.nwu.edu).

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific, being directed against a single epitope. The term “monoclonalantibody” encompasses not only intact monoclonal antibodies andfull-length monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)2, Fv), single chain (ScFv), variants thereof, fusionproteins comprising an antigen-binding portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals, etc.). The term includes whole immunoglobulins aswell as the fragments etc. described above under the definition of“antibody”.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)2 fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments of the present invention can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions of an IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

In certain embodiments, single chain Fv or scFV antibodies arecontemplated. For example, Kappa bodies (III et al., Prot. Eng. 10:949-57 (1997); minibodies (Martin et al., EMBO J 13: 5305-9 (1994);diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins(Traunecker et al., EMBO J 10: 3655-59 (1991) and Traunecker et al.,Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity. In still other embodiments, bispecific or chimericantibodies may be made that encompass the ligands of the presentdisclosure. For example, a chimeric antibody may comprise CDRs andframework regions from different antibodies, while bispecific antibodiesmay be generated that bind specifically to CD40 through one bindingdomain and to a second molecule through a second binding domain. Theseantibodies may be produced through recombinant molecular biologicaltechniques or may be physically conjugated together.

A single chain Fv (sFv) polypeptide is a covalently linked V_(H)::V_(L)heterodimer which is expressed from a gene fusion including V_(H)- andV_(L)-encoding genes linked by a peptide-encoding linker. Huston et al.(1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated—but chemically separated—light and heavypolypeptide chains from an antibody V region into an sFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

In certain embodiments, a CD40 binding antibody as described herein isin the form of a diabody. Diabodies are multimers of polypeptides, eachpolypeptide comprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

A dAb fragment of an antibody consists of a VH domain (Ward, E. S. etal., Nature 341, 544-546 (1989)).

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above. Diabodies andscFv can be constructed without an Fc region, using only variabledomains, potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against antigen X, then a library can be made where the otherarm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by knobs-into-holes engineering(J. B. B. Ridgeway et al., Protein Eng., 9, 616-621, 1996).

In certain embodiments, the antibodies described herein may be providedin the form of a UniBody®. A UniBody® is an IgG4 antibody with the hingeregion removed (see GenMab Utrecht, The Netherlands; see also, e.g.,US20090226421). This proprietary antibody technology creates a stable,smaller antibody format with an anticipated longer therapeutic windowthan current small antibody formats. IgG4 antibodies are consideredinert and thus do not interact with the immune system. Fully human IgG4antibodies may be modified by eliminating the hinge region of theantibody to obtain half-molecule fragments having distinct stabilityproperties relative to the corresponding intact IgG4 (GenMab, Utrecht).Halving the IgG4 molecule leaves only one area on the UniBody® that canbind to cognate antigens (e.g., disease targets) and the UniBody®therefore binds univalently to only one site on target cells. Forcertain cancer cell surface antigens, this univalent binding may notstimulate the cancer cells to grow as may be seen using bivalentantibodies having the same antigen specificity, and hence UniBody®technology may afford treatment options for some types of cancer thatmay be refractory to treatment with conventional antibodies. The smallsize of the UniBody® can be a great benefit when treating some forms ofcancer, allowing for better distribution of the molecule over largersolid tumors and potentially increasing efficacy.

In certain embodiments, the antibodies of the present disclosure maytake the form of a nanobody. Nanobodies are encoded by single genes andare efficiently produced in almost all prokaryotic and eukaryotic hostse.g. E. coli (see e.g. U.S. Pat. No. 6,765,087), moulds (for exampleAspergillus or Trichoderma) and yeast (for example Saccharomyces,Kluyvermyces, Hansenula or Pichia (see e.g. U.S. Pat. No. 6,838,254).The production process is scalable and multi-kilogram quantities ofnanobodies have been produced. Nanobodies may be formulated as aready-to-use solution having a long shelf life. The Nanoclone method(see, e.g., WO 06/079372) is a proprietary method for generatingNanobodies against a desired target, based on automated high-throughputselection of B-cells.

In certain embodiments, the anti-CD40 antibodies or antigen-bindingfragments thereof as disclosed herein are humanized. This refers to achimeric molecule, generally prepared using recombinant techniques,having an antigen-binding site derived from an immunoglobulin from anon-human species and the remaining immunoglobulin structure of themolecule based upon the structure and/or sequence of a humanimmunoglobulin. The antigen-binding site may comprise either completevariable domains fused onto constant domains or only the CDRs graftedonto appropriate framework regions in the variable domains. Epitopebinding sites may be wild type or modified by one or more amino acidsubstitutions. This eliminates the constant region as an immunogen inhuman individuals, but the possibility of an immune response to theforeign variable region remains (LoBuglio, A. F. et al., (1989) ProcNatl Acad Sci USA 86:4220-4224; Queen et al., PNAS (1988)86:10029-10033; Riechmann et al., Nature (1988) 332:323-327).Illustrative methods for humanization of the anti-CD40 antibodiesdisclosed herein include the methods described in U.S. Pat. No.7,462,697. Illustrative humanized antibodies according to certainembodiments of the present invention comprise the humanized sequencesprovided in SEQ ID NOs:9 and 10.

Another approach focuses not only on providing human-derived constantregions, but modifying the variable regions as well so as to reshapethem as closely as possible to human form. It is known that the variableregions of both heavy and light chains contain threecomplementarity-determining regions (CDRs) which vary in response to theepitopes in question and determine binding capability, flanked by fourframework regions (FRs) which are relatively conserved in a givenspecies and which putatively provide a scaffolding for the CDRs. Whennonhuman antibodies are prepared with respect to a particular epitope,the variable regions can be “reshaped” or “humanized” by grafting CDRsderived from nonhuman antibody on the FRs present in the human antibodyto be modified. Application of this approach to various antibodies hasbeen reported by Sato, K., et al., (1993) Cancer Res 53:851-856.Riechmann, L., et al., (1988) Nature 332:323-327; Verhoeyen, M., et al.,(1988) Science 239:1534-1536; Kettleborough, C. A., et al., (1991)Protein Engineering 4:773-3783; Maeda, H., et al., (1991) HumanAntibodies Hybridoma 2:124-134; Gorman, S. D., et al., (1991) Proc NatlAcad Sci USA 88:4181-4185; Tempest, P. R., et al., (1991) Bio/Technology9:266-271; Co, M. S., et al., (1991) Proc Natl Acad Sci USA88:2869-2873; Carter, P., et al., (1992) Proc Natl Acad Sci USA89:4285-4289; and Co, M. S. et al., (1992) J Immunol 148:1149-1154. Insome embodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

In certain embodiments, the antibodies of the present disclosure may bechimeric antibodies. In this regard, a chimeric antibody is comprised ofan antigen-binding fragment of an anti-CD40 antibody operably linked orotherwise fused to a heterologous Fc portion of a different antibody. Incertain embodiments, the heterologous Fc domain is of human origin. Inother embodiments, the heterologous Fc domain may be from a different Igclass from the parent antibody, including IgA (including subclasses IgA1and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, andIgG4), and IgM. In further embodiments, the heterologous Fc domain maybe comprised of CH2 and CH3 domains from one or more of the different Igclasses. As noted above with regard to humanized antibodies, theanti-CD40 antigen-binding fragment of a chimeric antibody may compriseonly one or more of the CDRs of the antibodies described herein (e.g.,1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or maycomprise an entire variable domain (VL, VH or both).

In certain embodiments, a CD40-binding antibody comprises one or more ofthe CDRs of the antibodies described herein. In this regard, it has beenshown in some cases that the transfer of only the VHCDR3 of an antibodycan be performed while still retaining desired specific binding (Barbaset al., PNAS (1995) 92: 2529-2533). See also, McLane et al., PNAS (1995)92:5214-5218, Barbas et al., J. Am. Chem. Soc. (1994) 116:2161-2162.

Marks et al (Bio/Technology, 1992, 10:779-783) describe methods ofproducing repertoires of antibody variable domains in which consensusprimers directed at or adjacent to the 5′ end of the variable domainarea are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VHvariable domains lacking a CDR3. Marks et al further describe how thisrepertoire may be combined with a CDR3 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentlydescribed antibodies may be shuffled with repertoires of VH or VLdomains lacking a CDR3, and the shuffled complete VH or VL domainscombined with a cognate VL or VH domain to provide an antibody orantigen-binding fragment thereof that binds CD40. The repertoire maythen be displayed in a suitable host system such as the phage displaysystem of WO92/01047 so that suitable antibodies or antigen-bindingfragments thereof may be selected. A repertoire may consist of at leastfrom about 10⁴ individual members and upwards by several orders ofmagnitude, for example, to about from 10⁶ to 10⁸ or 10¹⁰ or moremembers. Analogous shuffling or combinatorial techniques are alsodisclosed by Stemmer (Nature, 1994, 370:389-391), who describes thetechnique in relation to a β-lactamase gene but observes that theapproach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying oneor more CDR-derived sequences of the herein described inventionembodiments using random mutagenesis of one or more selected VH and/orVL genes to generate mutations within the entire variable domain. Such atechnique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA,89:3576-3580), who used error-prone PCR. Another method which may beused is to direct mutagenesis to CDR regions of VH or VL genes. Suchtechniques are disclosed by Barbas et al., (1994, Proc. Natl. Acad.Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol.263:551-567).

In certain embodiments, a specific VH and/or VL of the antibodiesdescribed herein may be used to screen a library of the complementaryvariable domain to identify antibodies with desirable properties, suchas increased affinity for CD40. Such methods are described, for example,in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al.,Nature (1991) 352:624-628.

Other methods may also be used to mix and match CDRs to identifyantibodies having desired binding activity, such as binding to CD40. Forexample: Klimka et al., British Journal of Cancer (2000) 83: 252-260,describe a screening process using a mouse VL and a human VH librarywith CDR3 and FR4 retained from the mouse VH. After obtainingantibodies, the VH was screened against a human VL library to obtainantibodies that bound antigen. Beiboer et al., J. Mol. Biol. (2000)296:833-849 describe a screening process using an entire mouse heavychain and a human light chain library. After obtaining antibodies, oneVL was combined with a human VH library with the CDR3 of the mouseretained. Antibodies capable of binding antigen were obtained. Rader etal., PNAS (1998) 95:8910-8915 describe a process similar to Beiboer etal above.

These just-described techniques are, in and of themselves, known as suchin the art. The skilled person will, however, be able to use suchtechniques to obtain antibodies or antigen-binding fragments thereofaccording to several embodiments of the invention described herein,using routine methodology in the art.

Also disclosed herein is a method for obtaining an antibody antigenbinding domain specific for CD40 antigen, the method comprisingproviding by way of addition, deletion, substitution or insertion of oneor more amino acids in the amino acid sequence of a VH domain set outherein a VH domain which is an amino acid sequence variant of the VHdomain, optionally combining the VH domain thus provided with one ormore VL domains, and testing the VH domain or VH/VL combination orcombinations to identify a specific binding member or an antibodyantigen binding domain specific for CD40 and optionally with one or moredesired properties. The VL domains may have an amino acid sequence whichis substantially as set out herein. An analogous method may be employedin which one or more sequence variants of a VL domain disclosed hereinare combined with one or more VH domains.

An epitope that “specifically binds” or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a CD40 epitope is an antibody that binds oneCD40 epitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other CD40 epitopes or non-CD40epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(d) represents a greater affinity.Immunological binding properties of selected polypeptides can bequantified using methods well known in the art. One such method entailsmeasuring the rates of antigen-binding site/antigen complex formationand dissociation, wherein those rates depend on the concentrations ofthe complex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (K_(on)) and the “off rate constant”(K_(off)) can be determined by calculation of the concentrations and theactual rates of association and dissociation. The ratio ofK_(off)/K_(on) enables cancellation of all parameters not related toaffinity, and is thus equal to the dissociation constant K_(d). See,generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

In certain embodiments, the anti-CD40 antibodies described herein havean affinity of about 100, 150, 155, 160, 170, 175, 180, 185, 190, 191,192, 193, 194, 195, 196, 197, 198 or 199 picomolar, and in someembodiments, the antibodies may have even higher affinity for CD40.

The term “immunologically active”, with reference to an epitope being or“remaining immunologically active”, refers to the ability of an antibody(e.g., anti-CD40 antibody) to bind to the epitope under differentconditions, for example, after the epitope has been subjected toreducing and denaturing conditions.

An antibody or antigen-binding fragment thereof according to certainpreferred embodiments of the present application may be one thatcompetes for binding to CD40 with any antibody described herein whichboth (i) specifically binds to the antigen and (ii) comprises a VHand/or VL domain disclosed herein, or comprises a VH CDR3 disclosedherein, or a variant of any of these. Competition between antibodies maybe assayed easily in vitro, for example using ELISA and/or by tagging aspecific reporter molecule to one antibody which can be detected in thepresence of other untagged antibodies, to enable identification ofspecific antibodies which bind the same epitope or an overlappingepitope. Thus, there is provided herein a specific antibody orantigen-binding fragment thereof, comprising a human antibodyantigen-binding site which competes with an antibody described hereinthat binds to CD40.

In this regard, as used herein, the terms “competes with”, “inhibitsbinding” and “blocks binding” (e.g., referring to inhibition/blocking ofbinding of CD40L to CD40 or referring to inhibition/blocking of bindingof an anti-CD40 antibody to CD40) are used interchangeably and encompassboth partial and complete inhibition/blocking. Inhibition and blockingare also intended to include any measurable decrease in the binding ofCD40L to CD40 when in contact with an anti-CD40 antibody as disclosedherein as compared to the ligand not in contact with an anti-CD40antibody, e.g., the blocking of CD40L to CD40 by at least about 10%,20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%.

The constant regions of immunoglobulins show less sequence diversitythan the variable regions, and are responsible for binding a number ofnatural proteins to elicit important biochemical events. In humans thereare five different classes of antibodies including IgA (which includessubclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclassesIgG1, IgG2, IgG3, and IgG4), and IgM. The distinguishing featuresbetween these antibody classes are their constant regions, althoughsubtler differences may exist in the V region.

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region comprises Igdomains CH2 and CH3 and the N-terminal hinge leading into CH2. Animportant family of Fc receptors for the IgG class are the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290). In humans this protein family includes FcγRI(CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32),including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb(including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16),including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferiset al., 2002, Immunol Lett 82:57-65). These receptors typically have anextracellular domain that mediates binding to Fc, a membrane spanningregion, and an intracellular domain that may mediate some signalingevent within the cell. These receptors are expressed in a variety ofimmune cells including monocytes, macrophages, neutrophils, dendriticcells, eosinophils, mast cells, platelets, B cells, large granularlymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells.Formation of the Fc/FcγR complex recruits these effector cells to sitesof bound antigen, typically resulting in signaling events within thecells and important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack.

The ability to mediate cytotoxic and phagocytic effector functions is apotential mechanism by which antibodies destroy targeted cells. Thecell-mediated reaction wherein nonspecific cytotoxic cells that expressFcγRs recognize bound antibody on a target cell and subsequently causelysis of the target cell is referred to as antibody dependentcell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev CellDev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766;Ravetch et al., 2001, Annu Rev Immunol 19:275-290). The cell-mediatedreaction wherein nonspecific cytotoxic cells that express FcγRsrecognize bound antibody on a target cell and subsequently causephagocytosis of the target cell is referred to as antibody dependentcell-mediated phagocytosis (ADCP). All FcγRs bind the same region on Fc,at the N-terminal end of the Cg2 (CH2) domain and the preceding hinge.This interaction is well characterized structurally (Sondermann et al.,2001, J Mol Biol 309:737-749), and several structures of the human Fcbound to the extracellular domain of human FcγRIIIb have been solved(pdb accession code 1 E4K)(Sondermann et al., 2000, Nature 406:267-273)(pdb accession codes 111 S and 1IIX)(Radaev et al., 2001, J Biol Chem276:16469-16477.)

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65). All FcγRs bind the same region on IgG Fc, yet with differentaffinities: the high affinity binder FcγRI has a K_(d) for IgG1 of 10⁻⁸M⁻¹, whereas the low affinity receptors FcγRII and FcγRIII generallybind at 10⁻⁶ and 10⁻⁵ respectively. The extracellular domains ofFcγRIIIa and FcγRIIIb are 96% identical, however FcγRIIIb does not havea intracellular signaling domain. Furthermore, whereas FcγRI, FcγRIIa/c,and FcγRIIIa are positive regulators of immune complex-triggeredactivation, characterized by having an intracellular domain that has animmunoreceptor tyrosine-based activation motif (ITAM), FcγRIIb has animmunoreceptor tyrosine-based inhibition motif (ITIM) and is thereforeinhibitory. Thus the former are referred to as activation receptors, andFcγRIIb is referred to as an inhibitory receptor. The receptors alsodiffer in expression pattern and levels on different immune cells. Yetanother level of complexity is the existence of a number of FcγRpolymorphisms in the human proteome. A particularly relevantpolymorphism with clinical significance is V158/F158 FcγRIIIa. HumanIgG1 binds with greater affinity to the V158 allotype than to the F158allotype. This difference in affinity, and presumably its effect on ADCCand/or ADCP, has been shown to be a significant determinant of theefficacy of the anti-CD20 antibody rituximab (Rituxan®, a registeredtrademark of IDEC Pharmaceuticals Corporation). Patients with the V158allotype respond favorably to rituximab treatment; however, patientswith the lower affinity F158 allotype respond poorly (Cartron et al.,2002, Blood 99:754-758). Approximately 10-20% of humans are V158/V158homozygous, 45% are V158/F158 heterozygous, and 35-45% of humans areF158/F158 homozygous (Lehrnbecher et al., 1999, Blood 94:4220-4232;Cartron et al., 2002, Blood 99:754-758). Thus 80-90% of humans are poorresponders, that is they have at least one allele of the F158 FcγRIIIa.

The Fc region is also involved in activation of the complement cascade.In the classical complement pathway, C1 binds with its C1q subunits toFc fragments of IgG or IgM, which has formed a complex with antigen(s).In certain embodiments of the invention, modifications to the Fc regioncomprise modifications that alter (either enhance or decrease) theability of a CD40-specific antibody as described herein to activate thecomplement system (see e.g., U.S. Pat. No. 7,740,847). To assesscomplement activation, a complement-dependent cytotoxicity (CDC) assaymay be performed (See, e.g., Gazzano-Santoro et al., J. Immunol.Methods, 202:163 (1996)).

Thus in certain embodiments, the present invention provides anti-CD40antibodies having a modified Fc region with altered functionalproperties, such as reduced or enhanced CDC, ADCC, or ADCP activity, orenhanced binding affinity for a specific FcγR or increased serumhalf-life. Other modified Fc regions contemplated herein are described,for example, in issued U.S. Pat. Nos. 7,317,091; 7,657,380; 7,662,925;6,538,124; 6,528,624; 7,297,775; 7,364,731; Published U.S. ApplicationsUS2009092599; US20080131435; US20080138344; and published InternationalApplications WO2006/105338; WO2004/063351; WO2006/088494; WO2007/024249.

In one embodiment, one or more substitutions in the Fc may increase thebinding affinity to FcγRIIB, enhance crosslinking of CD40 molecules andlead to stronger CD40 activation by an anti-CD40 antibody. In oneembodiment, the present invention provides anti-CD40 antibodies having amodified Fc region at position 267 (EU numbering; see e.g., Edelman, G.M. et al., 1969 Proc. Natl. Acad. USA, 63, 78-85; see also theImMunoGeneTics (IMGT) database website atimgt.org/IMGTScientificChart/Numbering). In one embodiment, an anti-CD40antibody herein comprises a modified Fc comprising a S267E substitution(Li Fu, Ravetch J V. 2011 Science 333:1030; see also J. Immunol. 2011,187:1754-1763; mAbs 2010, 2:181-189). The amino acid sequence of a heavychain constant region comprising an illustrative modified Fc is setforth in SEQ ID NO:195.

Thus, in certain embodiments, antibody variable domains with the desiredbinding specificities are fused to immunoglobulin constant domainsequences. In certain embodiments, the fusion is with an Ig heavy chainconstant domain, comprising at least part of the hinge, C_(H)2, andC_(H)3 regions. It is preferred to have the first heavy-chain constantregion (C_(H)1) containing the site necessary for light chain bonding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host cell. This provides for greater flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yield of the desired bispecificantibody. It is, however, possible to insert the coding sequences fortwo or all three polypeptide chains into a single expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios have no significant affect onthe yield of the desired chain combination.

Antibodies of the present invention (and antigen-binding fragments andvariants thereof) may also be modified to include an epitope tag orlabel, e.g., for use in purification or diagnostic applications. Thereare many linking groups known in the art for making antibody conjugates,including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EPPatent 0 425 235 B1, and Chari et al., Cancer Research 52: 127-131(1992). The linking groups include disufide groups, thioether groups,acid labile groups, photolabile groups, peptidase labile groups, oresterase labile groups, as disclosed in the above-identified patents,disulfide and thioether groups being preferred.

In another contemplated embodiment, a CD40-specific antibody asdescribed herein may be conjugated or operably linked to anothertherapeutic compound, referred to herein as a conjugate. The conjugatemay be a cytotoxic agent, a chemotherapeutic agent, a cytokine, ananti-angiogenic agent, a tyrosine kinase inhibitor, a toxin, aradioisotope, or other therapeutically active agent. Chemotherapeuticagents, cytokines, anti-angiogenic agents, tyrosine kinase inhibitors,and other therapeutic agents have been described above, and all of theseaforemention therapeutic agents may find use as antibody conjugates.

In an alternate embodiment, the antibody is conjugated or operablylinked to a toxin, including but not limited to small molecule toxinsand enzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof. Small moleculetoxins include but are not limited to saporin (Kuroda K, et al., TheProstate 70:1286-1294 (2010); Lip, W L. et al., 2007 MolecularPharmaceutics 4:241-251; Quadros E V., et al., 2010 Mol Cancer Ther;9(11); 3033-40; Polito L., et al. 2009 British Journal of Haematology,147, 710-718), calicheamicin, maytansine (U.S. Pat. No. 5,208,020),trichothene, and CC1065. Toxins include but are not limited to RNase,gelonin, enediynes, ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin (PE40), Shigella toxin, Clostridiumperfringens toxin, and pokeweed antiviral protein.

In one embodiment, an antibody or antigen-binding fragment thereof ofthe disclosure is conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors that act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

Another conjugate of interest comprises an antibody conjugated to one ormore calicheamicin molecules. The calicheamicin family of antibioticsare capable of producing double-stranded DNA breaks at sub-picomolarconcentrations. Structural analogues of calicheamicin that may also beused (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,1998, Cancer Research 58:2925-2928) (U.S. Pat. No. 5,714,586; U.S. Pat.No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No. 5,773,001).Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatinE (MMAE) may find use as conjugates for the presently disclosedantibodies, or variants thereof (Doronina et al., 2003, Nat Biotechnol21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65). Usefulenzymatically active toxins include but are not limited to diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, enomycin and thetricothecenes. See, for example, PCT WO 93/21232. The present disclosurefurther contemplates embodiments in which a conjugate or fusion isformed between a CD40-specific antibody as described herein and acompound with nucleolytic activity, for example a ribonuclease or DNAendonuclease such as a deoxyribonuclease (DNase).

In an alternate embodiment, a herein-disclosed antibody may beconjugated or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and ²¹²Bi.

Antibodies described herein may in certain other embodiments beconjugated to a therapeutic moiety such as a cytotoxin (e.g., acytostatic or cytocidal agent), a therapeutic agent or a radioactiveelement (e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins orcytotoxic agents include any agent that is detrimental to cells.Examples include paclitaxel/paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Onepreferred exemplary cytotoxin is saporin (available from AdvancedTargeting Systems, San Diego, Calif.). Therapeutic agents include, butare not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, a CD40-specific antibody (including a functional fragmentthereof as provided herein such as an antigen-binding fragment) may incertain embodiments be conjugated to therapeutic moieties such as aradioactive materials or macrocyclic chelators useful for conjugatingradiometal ions. In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide). In an alternateembodiment, the antibody is conjugated or operably linked to an enzymein order to employ Antibody Dependent Enzyme Mediated Prodrug Therapy(ADEPT). ADEPT may be used by conjugating or operably linking theantibody to a prodrug-activating enzyme that converts a prodrug (e.g. apeptidyl chemotherapeutic agent, see PCT WO 81/01145) to an activeanti-cancer drug. See, for example, PCT WO 88/07378 and U.S. Pat. No.4,975,278. The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto convert it into its more active, cytotoxic form. Enzymes that areuseful in the method of these and related embodiments include but arenot limited to alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as □-galactosidase and neuramimidaseuseful for converting glycosylated prodrugs into free drugs;beta-lactamase useful for converting drugs derivatized with □-lactamsinto free drugs; and penicillin amidases, such as penicillin V amidaseor penicillin G amidase, useful for converting drugs derivatized attheir amine nitrogens with phenoxyacetyl or phenylacetyl groups,respectively, into free drugs. Alternatively, antibodies with enzymaticactivity, also known in the art as “abzymes”, may be used to convertprodrugs into free active drugs (see, for example, Massey, 1987, Nature328: 457-458). Antibody-abzyme conjugates can be prepared for deliveryof the abzyme to a tumor cell population.

Immunoconjugates may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP), succinim idyl-4-(N-maleim idomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particular coupling agents includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al.,Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage. The linker may be a “cleavable linker” facilitatingrelease of one or more cleavable components. For example, an acid-labilelinker may be used (Cancer Research 52: 127-131 (1992); U.S. Pat. No.5,208,020).

Other modifications of the antibodies (and polypeptides) of theinvention are also contemplated herein. For example, the antibody may belinked to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol. The antibodyalso may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate)microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN™)polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.

As noted elsewhere herein, the antibodies of the present disclosureinduce CD40 signalling in tumor cells, activate dendritic cells andimmune surveillance, activate antibody dependent cellular cytotoxicity(ADCC) against tumor cells, block binding of CD40 to CD40L; have CD40agonistic activity; activate antigen presenting cells; stimulatecytokine release from antigen presenting cells; induce tumor cellapoptosis; inhibit tumor cell proliferation; kill tumor cells viainduction of effector functions including but not limited to ADCC, CDCand ADCP; stimulate anti-tumor T cell responses; reduce establishedtumors; and inhibit rituximab-resistant tumors. The antibodies describedherein may have or induce a combination of any one or more of theseattributes or activities. The desired functional properties of anti-CD40antibodies may be assessed using a variety of methods known to theskilled person, such as affinity/binding assays (for example, surfaceplasmon resonance, competitive inhibition assays); cytotoxicity assays,cell viability assays, cell proliferation, activation or differentiationassays, ADCC and CDC assays, other cellular activity resulting from CD40cell signalling events (e.g., STAT3 phosporylation, production ofcytokines including IL-1, IL-6, IL-8, IL-10, IL-12, TNF-Alpha, andMIP1AIpha), and cancer cell and/or tumor growth inhibition using invitro or in vivo models. Other assays may test the ability of antibodiesdescribed herein to block normal CD40L binding to CD40 or CD40-mediatedresponses, such as cell signalling, cell activation (e.g., immune cellactivation, proliferation; antigen presenting cell activation (e.g.,dendritic cells, B cells, macrophages) and maturation assays), immuneresponses (including cell mediated and humoral responses), etc. Theantibodies described herein may also be tested for effects on CD40internalisation, in vitro and in vivo efficacy, etc. Such assays may beperformed using well-established protocols known to the skilled person(see e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc.Inc. & John Wiley & Sons, Inc., NY, NY); Current Protocols in Immunology(Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, EthanM. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); orcommercially available kits.

The present invention further provides in certain embodiments anisolated nucleic acid encoding an antibody or antigen-binding fragmentthereof as described herein, for instance, a nucleic acid which codesfor a CDR or VH or VL domain as described herein. Nucleic acids includeDNA and RNA. These and related embodiments may include polynucleotidesencoding antibodies that bind CD40 as described herein. The term“isolated polynucleotide” as used herein shall mean a polynucleotide ofgenomic, cDNA, or synthetic origin or some combination thereof, which byvirtue of its origin the isolated polynucleotide (1) is not associatedwith all or a portion of a polynucleotide in which the isolatedpolynucleotide is found in nature, (2) is linked to a polynucleotide towhich it is not linked in nature, or (3) does not occur in nature aspart of a larger sequence.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, atranscription control sequence “operably linked” to a protein codingsequence is ligated thereto so that expression of the protein codingsequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can affect expression, processing or intracellularlocalization of coding sequences to which they are ligated or operablylinked. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, transcription control sequences forprokaryotes may include a promoter, ribosomal binding site, andtranscription termination sequence. In other particular embodiments,transcription control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences, transcription terminationsequences and polyadenylation sequences. In certain embodiments,“control sequences” can include leader sequences and/or fusion partnersequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers. In certain embodiments, thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine,ribose modifications such as arabinoside and 2′,3′-dideoxyribose andinternucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term“polynucleotide” specifically includes single and double stranded formsof DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control expression of inserted heterologous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.

As will be understood by those skilled in the art, polynucleotides mayinclude genomic sequences, extra-genomic and plasmid-encoded sequencesand smaller engineered gene segments that express, or may be adapted toexpress, proteins, polypeptides, peptides and the like. Such segmentsmay be naturally isolated, or modified synthetically by the skilledperson.

As will be also recognized by the skilled artisan, polynucleotides maybe single-stranded (coding or antisense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules mayinclude HnRNA molecules, which contain introns and correspond to a DNAmolecule in a one-to-one manner, and mRNA molecules, which do notcontain introns. Additional coding or non-coding sequences may, but neednot, be present within a polynucleotide according to the presentdisclosure, and a polynucleotide may, but need not, be linked to othermolecules and/or support materials. Polynucleotides may comprise anative sequence or may comprise a sequence that encodes a variant orderivative of such a sequence.

Therefore, according to these and related embodiments, the presentdisclosure also provides polynucleotides encoding the anti-CD40antibodies described herein. In certain embodiments, polynucleotides areprovided that comprise some or all of a polynucleotide sequence encodingan antibody as described herein and complements of such polynucleotides.

In other related embodiments, polynucleotide variants may havesubstantial identity to a polynucleotide sequence encoding an anti-CD40antibody described herein. For example, a polynucleotide may be apolynucleotide comprising at least 70% sequence identity, preferably atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequenceidentity compared to a reference polynucleotide sequence such as asequence encoding an antibody described herein, using the methodsdescribed herein, (e.g., BLAST analysis using standard parameters, asdescribed below). One skilled in this art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the binding affinity of the antibody encoded by the variantpolynucleotide is not substantially diminished relative to an antibodyencoded by a polynucleotide sequence specifically set forth herein.

In certain other related embodiments, polynucleotide fragments maycomprise or consist essentially of various lengths of contiguousstretches of sequence identical to or complementary to a sequenceencoding an antibody as described herein. For example, polynucleotidesare provided that comprise or consist essentially of at least about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200,300, 400, 500 or 1000 or more contiguous nucleotides of a sequences theencodes an antibody, or antigen-binding fragment thereof, disclosedherein as well as all intermediate lengths there between. It will bereadily understood that “intermediate lengths”, in this context, meansany length between the quoted values, such as 50, 51, 52, 53, etc.; 100,101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integersthrough 200-500; 500-1,000, and the like. A polynucleotide sequence asdescribed here may be extended at one or both ends by additionalnucleotides not found in the native sequence. This additional sequencemay consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 nucleotides at either end of a polynucleotide encodingan antibody described herein or at both ends of a polynucleotideencoding an antibody described herein.

In another embodiment, polynucleotides are provided that are capable ofhybridizing under moderate to high stringency conditions to apolynucleotide sequence encoding an antibody, or antigen-bindingfragment thereof, provided herein, or a fragment thereof, or acomplementary sequence thereof. Hybridization techniques are well knownin the art of molecular biology. For purposes of illustration, suitablemoderately stringent conditions for testing the hybridization of apolynucleotide as provided herein with other polynucleotides includeprewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at 50° C.-60° C., 5×SSC, overnight; followed by washingtwice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. One skilled in the art will understand that thestringency of hybridization can be readily manipulated, such as byaltering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60° C.-65° C. or 65° C.-70° C.

In certain embodiments, the polynucleotides described above, e.g.,polynucleotide variants, fragments and hybridizing sequences, encodeantibodies that bind CD40, or antigen-binding fragments thereof. Inother embodiments, such polynucleotides encode antibodies orantigen-binding fragments, or CDRs thereof, that bind to CD40 at leastabout 50%, at least about 70%, and in certain embodiments, at leastabout 90% as well as an antibody sequence specifically set forth herein.In further embodiments, such polynucleotides encode antibodies orantigen-binding fragments, or CDRs thereof, that bind to CD40 withgreater affinity than the antibodies set forth herein, for example, thatbind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110%as well as an antibody sequence specifically set forth herein.

As described elsewhere herein, determination of the three-dimensionalstructures of representative polypeptides (e.g., variant CD40-specificantibodies as provided herein, for instance, an antibody protein havingan antigen-binding fragment as provided herein) may be made throughroutine methodologies such that substitution, addition, deletion orinsertion of one or more amino acids with selected natural ornon-natural amino acids can be virtually modeled for purposes ofdetermining whether a so derived structural variant retains thespace-filling properties of presently disclosed species. A variety ofcomputer programs are known to the skilled artisan for determiningappropriate amino acid substitutions (or appropriate polynucleotidesencoding the amino acid sequence) within an antibody such that, forexample, affinity is maintained or better affinity is achieved.

The polynucleotides described herein, or fragments thereof, regardlessof the length of the coding sequence itself, may be combined with otherDNA sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, illustrative polynucleotide segments with totallengths of about 10,000, about 5000, about 3000, about 2,000, about1,000, about 500, about 200, about 100, about 50 base pairs in length,and the like, (including all intermediate lengths) are contemplated tobe useful.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity among two or more the polynucleotides. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

In certain embodiments, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) of 20 percent or less, usually 5 to 15percent, or 10 to 12 percent, as compared to the reference sequences(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The percentage is calculated by determining thenumber of positions at which the identical nucleic acid bases occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thereference sequence (i.e., the window size) and multiplying the resultsby 100 to yield the percentage of sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode an antibody as described herein. Some of thesepolynucleotides bear minimal sequence identity to the nucleotidesequence of the native or original polynucleotide sequence that encodeantibodies that bind to CD40. Nonetheless, polynucleotides that vary dueto differences in codon usage are expressly contemplated by the presentdisclosure. In certain embodiments, sequences that have beencodon-optimized for mammalian expression are specifically contemplated.

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, may be employed for thepreparation of variants and/or derivatives of the antibodies describedherein. By this approach, specific modifications in a polypeptidesequence can be made through mutagenesis of the underlyingpolynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate the mutagenesis of thepolynucleotide sequences that encode an antibody disclosed herein, or anantigen-binding fragment thereof, to alter one or more properties of theencoded polypeptide, such as the binding affinity of the antibody or theantigen-binding fragment thereof, or the function of a particular Fcregion, or the affinity of the Fc region for a particular FcγR. Thetechniques of site-specific mutagenesis are well-known in the art, andare widely used to create variants of both polypeptides andpolynucleotides. For example, site-specific mutagenesis is often used toalter a specific portion of a DNA molecule. In such embodiments, aprimer comprising typically about 14 to about 25 nucleotides or so inlength is employed, with about 5 to about 10 residues on both sides ofthe junction of the sequence being altered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants,recursive sequence recombination, as described in U.S. Pat. No.5,837,458, may be employed. In this approach, iterative cycles ofrecombination and screening or selection are performed to “evolve”individual polynucleotide variants having, for example, increasedbinding affinity. Certain embodiments also provide constructs in theform of plasmids, vectors, transcription or expression cassettes whichcomprise at least one polynucleotide as described herein.

In many embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cellincubated under conditions sufficient to induce expression of theencoded antibody. The antibodies of this disclosure are prepared usingstandard techniques well known to those of skill in the art incombination with the polypeptide and nucleic acid sequences providedherein. The polypeptide sequences may be used to determine appropriatenucleic acid sequences encoding the particular antibody disclosedthereby. The nucleic acid sequence may be optimized to reflectparticular codon “preferences” for various expression systems accordingto standard methods well known to those of skill in the art.

According to certain related embodiments there is provided a recombinanthost cell which comprises one or more constructs as described herein; anucleic acid encoding any antibody, CDR, VH or VL domain, orantigen-binding fragment thereof; and a method of production of theencoded product, which method comprises expression from encoding nucleicacid therefor. Expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression, an antibody orantigen-binding fragment thereof, may be isolated and/or purified usingany suitable technique, and then used as desired.

Antibodies or antigen-binding fragments thereof as provided herein, andencoding nucleic acid molecules and vectors, may be isolated and/orpurified, e.g. from their natural environment, in substantially pure orhomogeneous form, or, in the case of nucleic acid, free or substantiallyfree of nucleic acid or genes of origin other than the sequence encodinga polypeptide with the desired function. Nucleic acid may comprise DNAor RNA and may be wholly or partially synthetic. Reference to anucleotide sequence as set out herein encompasses a DNA molecule withthe specified sequence, and encompasses a RNA molecule with thespecified sequence in which U is substituted for T, unless contextrequires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antigen-binding fragments inprokaryotic cells such as E. coli is well established in the art. For areview, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of antibodies orantigen-binding fragments thereof, see recent reviews, for example Ref,M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al.(1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992,or subsequent updates thereto.

The term “host cell” is used to refer to a cell into which has beenintroduced, or which is capable of having introduced into it, a nucleicacid sequence encoding one or more of the herein described antibodies,and which further expresses or is capable of expressing a selected geneof interest, such as a gene encoding any herein described antibody. Theterm includes the progeny of the parent cell, whether or not the progenyare identical in morphology or in genetic make-up to the originalparent, so long as the selected gene is present. Accordingly there isalso contemplated a method comprising introducing such nucleic acid intoa host cell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. The introductionmay be followed by causing or allowing expression from the nucleic acid,e.g. by culturing host cells under conditions for expression of thegene. In one embodiment, the nucleic acid is integrated into the genome(e.g. chromosome) of the host cell. Integration may be promoted byinclusion of sequences which promote recombination with the genome, inaccordance-with standard techniques.

The present invention also provides, in certain embodiments, a methodwhich comprises using a construct as stated above in an expressionsystem in order to express a particular polypeptide such as aCD40-specific antibody as described herein. The term “transduction” isused to refer to the transfer of genes from one bacterium to another,usually by a phage. “Transduction” also refers to the acquisition andtransfer of eukaryotic cellular sequences by retroviruses. The term“transfection” is used to refer to the uptake of foreign or exogenousDNA by a cell, and a cell has been “transfected” when the exogenous DNAhas been introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories;Davis et al., 1986, BASIC METHODS 1N MOLECULAR BIOLOGY, Elsevier; andChu et al., 1981, Gene 13:197. Such techniques can be used to introduceone or more exogenous DNA moieties into suitable host cells.

The term “transformation” as used herein refers to a change in a cell'sgenetic characteristics, and a cell has been transformed when it hasbeen modified to contain a new DNA. For example, a cell is transformedwhere it is genetically modified from its native state. Followingtransfection or transduction, the transforming DNA may recombine withthat of the cell by physically integrating into a chromosome of thecell, or may be maintained transiently as an episomal element withoutbeing replicated, or may replicate independently as a plasmid. A cell isconsidered to have been stably transformed when the DNA is replicatedwith the division of the cell. The term “naturally occurring” or“native” when used in connection with biological materials such asnucleic acid molecules, polypeptides, host cells, and the like, refersto materials which are found in nature and are not manipulated by ahuman. Similarly, “non-naturally occurring” or “non-native” as usedherein refers to a material that is not found in nature or that has beenstructurally modified or synthesized by a human.

The terms “polypeptide” “protein” and “peptide” and “glycoprotein” areused interchangeably and mean a polymer of amino acids not limited toany particular length. The term does not exclude modifications such asmyristylation, sulfation, glycosylation, phosphorylation and addition ordeletion of signal sequences. The terms “polypeptide” or “protein” meansone or more chains of amino acids, wherein each chain comprises aminoacids covalently linked by peptide bonds, and wherein said polypeptideor protein can comprise a plurality of chains non-covalently and/orcovalently linked together by peptide bonds, having the sequence ofnative proteins, that is, proteins produced by naturally-occurring andspecifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass theantibodies that bind to CD40 of the present disclosure, or sequencesthat have deletions from, additions to, and/or substitutions of one ormore amino acid of an anti-CD40 antibody. Thus, a “polypeptide” or a“protein” can comprise one (termed “a monomer”) or a plurality (termed“a multimer”) of amino acid chains.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldtypically be found in nature, (2) is essentially free of other proteinsfrom the same source, e.g., from the same species, (3) is expressed by acell from a different species, (4) has been separated from at leastabout 50 percent of polynucleotides, lipids, carbohydrates, or othermaterials with which it is associated in nature, (5) is not associated(by covalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of may be of synthetic origin, or any combinationthereof. In certain embodiments, the isolated protein is substantiallyfree from proteins or polypeptides or other contaminants that are foundin its natural environment that would interfere with its use(therapeutic, diagnostic, prophylactic, research or otherwise).

The term “polypeptide fragment” refers to a polypeptide, which can bemonomeric or multimeric, that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion or substitutionof a naturally-occurring or recombinantly-produced polypeptide. Incertain embodiments, a polypeptide fragment can comprise an amino acidchain at least 5 to about 500 amino acids long. It will be appreciatedthat in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200,250, 300, 350, 400, or 450 amino acids long. Particularly usefulpolypeptide fragments include functional domains, includingantigen-binding domains or fragments of antibodies. In the case of ananti-CD40 antibody, useful fragments include, but are not limited to: aCDR region, especially a CDR3 region of the heavy or light chain; avariable region of a heavy or light chain; a portion of an antibodychain or just its variable region including two CDRs; and the like.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. Any polypeptideamino acid sequences provided herein that include a signal peptide arealso contemplated for any use described herein without such a signal orleader peptide. As would be recognized by the skilled person, the signalpeptide is usually cleaved during processing and is not included in theactive antibody protein. The polypeptide may also be fused in-frame orconjugated to a linker or other sequence for ease of synthesis,purification or identification of the polypeptide (e.g., poly-His), orto enhance binding of the polypeptide to a solid support.

A peptide linker/spacer sequence may also be employed to separatemultiple polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and/or tertiary structures, ifdesired. Such a peptide linker sequence can be incorporated into afusion polypeptide using standard techniques well known in the art.

Certain peptide spacer sequences may be chosen, for example, based on:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and/or (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes.

In one illustrative embodiment, peptide spacer sequences contain, forexample, Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala, may also be included in the spacer sequence.

Other amino acid sequences which may be usefully employed as spacersinclude those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphyet al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No.4,935,233 and U.S. Pat. No. 4,751,180.

Other illustrative spacers may include, for example,Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary etal., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) andLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp(Bird et al., 1988, Science 242:423-426).

In some embodiments, spacer sequences are not required when the firstand second polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference. Two coding sequences can be fused directly without anyspacer or by using a flexible polylinker composed, for example, of thepentamer Gly-Gly-Gly-Gly-Ser repeated 1 to 3 times. Such a spacer hasbeen used in constructing single chain antibodies (scFv) by beinginserted between VH and VL (Bird et al., 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).

A peptide spacer, in certain embodiments, is designed to enable thecorrect interaction between two beta-sheets forming the variable regionof the single chain antibody.

In certain illustrative embodiments, a peptide spacer is between 1 to 5amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids,between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to50 amino acids, between 10 to 100 amino acids, or any intervening rangeof amino acids.

In other illustrative embodiments, a peptide spacer comprises about 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody. Forexample, amino acid sequence variants of an antibody may be prepared byintroducing appropriate nucleotide changes into a polynucleotide thatencodes the antibody, or a chain thereof, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution may be made to arrive at the final antibody, provided thatthe final construct possesses the desired characteristics (e.g., highaffinity binding to CD40). The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites. Any of the variations andmodifications described above for polypeptides of the present inventionmay be included in antibodies of the present invention.

The present disclosure provides variants of the antibodies disclosedherein. In certain embodiments, such variant antibodies orantigen-binding fragments, or CDRs thereof, bind to CD40 at least about50%, at least about 70%, and in certain embodiments, at least about 90%as well as an antibody sequence specifically set forth herein. Infurther embodiments, such variant antibodies or antigen-bindingfragments, or CDRs thereof, bind to CD40 with greater affinity than theantibodies set forth herein, for example, that bind quantitatively atleast about 105%, 106%, 107%, 108%, 109%, or 110% as well as an antibodysequence specifically set forth herein.

In particular embodiments, a subject antibody may have: a) a heavy chainvariable region having an amino acid sequence that is at least 80%identical, at least 95% identical, at least 90%, at least 95% or atleast 98% or 99% identical, to the heavy chain variable region of ananti-CD40 antibody described herein; and b) a light chain variableregion having an amino acid sequence that is at least 80% identical, atleast 85%, at least 90%, at least 95% or at least 98% or 99% identical,to the light chain variable region of an anti-CD40 antibody describedherein. The amino acid sequence of illustrative heavy and light chainregions are set forth in SEQ ID NOs:1-56.

In particular embodiments, the antibody may comprise: a) a heavy chainvariable region comprising: i. a CDR1 region that is identical in aminoacid sequence to the heavy chain CDR1 region of a selected antibodydescribed herein; ii. a CDR2 region that is identical in amino acidsequence to the heavy chain CDR2 region of the selected antibody; andiii. a CDR3 region that is identical in amino acid sequence to the heavychain CDR3 region of the selected antibody; and b) a light chainvariable domain comprising: i. a CDR1 region that is identical in aminoacid sequence to the light chain CDR1 region of the selected antibody;ii. a CDR2 region that is identical in amino acid sequence to the lightchain CDR2 region of the selected antibody; and iii. a CDR3 region thatis identical in amino acid sequence to the light chain CDR3 region ofthe selected antibody; wherein the antibody specifically binds aselected target (e.g., CD40). In a further embodiment, the antibody, orantigen-binding fragment thereof, is a variant antibody wherein thevariant comprises a heavy and light chain identical to the selectedantibody except for up to 8, 9, 10, 11, 12, 13, 14, 15, or more aminoacid substitutions in the CDR regions of the VH and VL regions. In thisregard, there may be 1, 2, 3, 4, 5, 6, 7, 8, or in certain embodiments,9, 10, 11, 12, 13, 14, 15 more amino acid substitutions in the CDRregions of the selected antibody. Substitutions may be in CDRs either inthe VH and/or the VL regions. (See e.g., Muller, 1998, Structure6:1153-1167).

Determination of the three-dimensional structures of representativepolypeptides (e.g., variant CD40-specific antibodies as provided herein,for instance, an antibody protein having an antigen-binding fragment asprovided herein) may be made through routine methodologies such thatsubstitution, addition, deletion or insertion of one or more amino acidswith selected natural or non-natural amino acids can be virtuallymodeled for purposes of determining whether a so derived structuralvariant retains the space-filling properties of presently disclosedspecies. See, for instance, Donate et al., 1994 Prot. Sci. 3:2378;Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al.,Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244(2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature450:259 (2007); Raman et al. Science 327:1014-1018 (2010). Someadditional non-limiting examples of computer algorithms that may be usedfor these and related embodiments, such as for rational design ofCD40-specific antibodies antigen-binding domains thereof as providedherein, include VMD which is a molecular visualization program fordisplaying, animating, and analyzing large biomolecular systems using3-D graphics and built-in scripting (see the website for the Theoreticaland Computational Biophysics Group, University of Illinois atUrbana-Champagne, at ks.uiuc.edu/Research/vmd/. Many other computerprograms are known in the art and available to the skilled person andwhich allow for determining atomic dimensions from space-filling models(van der Waals radii) of energy-minimized conformations; GRID, whichseeks to determine regions of high affinity for different chemicalgroups, thereby enhancing binding, Monte Carlo searches, which calculatemathematical alignment, and CHARMM (Brooks et al. (1983) J. Comput.Chem. 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106:765), which assess force field calculations, and analysis (see also,Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J.Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbamet al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. HealthPerspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn.9:475-488). A variety of appropriate computational computer programs arealso commercially available, such as from Schrödinger (Munich, Germany).

In another embodiment of invention, the anti-CD40 antibodies andhumanized versions thereof are derived from rabbit monoclonalantibodies, and in particular are generated using RabMAb® technology.These antibodies are advantageous as they require minimal sequencemodifications, thereby facilitating retention of functional propertiesafter humanization using mutational lineage guided (MLG) humanizationtechnology (see e.g., U.S. Pat. No. 7,462,697). Thus, illustrativemethods for making the anti-CD40 antibodies of the present disclosureinclude the RabMab® rabbit monoclonal antibody technology described, forexample, in U.S. Pat. Nos. 5,675,063 and 7,429,487. In this regard, incertain embodiments, the anti-CD40 antibodies of the disclosure areproduced in rabbits. In particular embodiments, a rabbit-derivedimmortal B-lymphocyte capable of fusion with a rabbit splenocyte is usedto produce a hybrid cell that produces an antibody. The immortalB-lymphocyte does not detectably express endogenous immunoglobulin heavychain and may contain, in certain embodiments, an altered immunoglobulinheavy chain-encoding gene.

Compositions and Methods of Use

The present disclosure provides compositions comprising theCD40-specific antibodies, antigen-binding fragments thereof andadministration of such composition in a variety of therapeutic settings.

Administration of the CD40-specific antibodies described herein, in pureform or in an appropriate pharmaceutical composition, can be carried outvia any of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions can be prepared bycombining an antibody or antibody-containing composition with anappropriate physiologically acceptable carrier, diluent or excipient,and may be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. In addition, other pharmaceutically active ingredients(including other anti-cancer agents as described elsewhere herein)and/or suitable excipients such as salts, buffers and stabilizers may,but need not, be present within the composition. Administration may beachieved by a variety of different routes, including oral, parenteral,nasal, intravenous, intradermal, subcutaneous or topical. Preferredmodes of administration depend upon the nature of the condition to betreated or prevented. An amount that, following administration, reduces,inhibits, prevents or delays the progression and/or metastasis of acancer is considered effective.

In certain embodiments, the amount administered is sufficient to resultin tumor regression, as indicated by a statistically significantdecrease in the amount of viable tumor, for example, at least a 50%decrease in tumor mass, or by altered (e.g., decreased with statisticalsignificance) scan dimensions. In other embodiments, the amountadministered is sufficient to result in clinically relevant reduction insymptoms of a particular disease indication known to the skilledclinician.

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

The CD40-specific antibody-containing compositions may be administeredalone or in combination with other known cancer treatments, such asradiation therapy, chemotherapy, transplantation, immunotherapy, hormonetherapy, photodynamic therapy, etc. The compositions may also beadministered in combination with antibiotics.

Typical routes of administering these and related pharmaceuticalcompositions thus include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Pharmaceutical compositions accordingto certain embodiments of the present invention are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a herein described CD40-specific antibody in aerosolform may hold a plurality of dosage units. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthis art; for example, see Remington: The Science and Practice ofPharmacy, 20th Edition (Philadelphia College of Pharmacy and Science,2000). The composition to be administered will, in any event, contain atherapeutically effective amount of an antibody of the presentdisclosure, for treatment of a disease or condition of interest inaccordance with teachings herein.

A pharmaceutical composition may be in the form of a solid or liquid. Inone embodiment, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral oil,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration. When intended for oral administration, thepharmaceutical composition is preferably in either solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of a CD40-specific antibodyas herein disclosed such that a suitable dosage will be obtained.Typically, this amount is at least 0.01% of the antibody in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Certain oral pharmaceutical compositions contain between about 4% andabout 75% of the antibody. In certain embodiments, pharmaceuticalcompositions and preparations according to the present invention areprepared so that a parenteral dosage unit contains between 0.01 to 10%by weight of the antibody prior to dilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration.

If intended for transdermal administration, the composition may includea transdermal patch or iontophoresis device. The pharmaceuticalcomposition may be intended for rectal administration, in the form, forexample, of a suppository, which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule. The pharmaceutical composition insolid or liquid form may include an agent that binds to the antibody ofthe invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include other monoclonalor polyclonal antibodies, one or more proteins or a liposome. Thepharmaceutical composition may consist essentially of dosage units thatcan be administered as an aerosol. The term aerosol is used to denote avariety of systems ranging from those of colloidal nature to systemsconsisting of pressurized packages. Delivery may be by a liquefied orcompressed gas or by a suitable pump system that dispenses the activeingredients. Aerosols may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient(s).Delivery of the aerosol includes the necessary container, activators,valves, subcontainers, and the like, which together may form a kit. Oneof ordinary skill in the art, without undue experimentation maydetermine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a composition that comprises a CD40-specific antibody asdescribed herein and optionally, one or more of salts, buffers and/orstabilizers, with sterile, distilled water so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the antibody composition so as to facilitate dissolutionor homogeneous suspension of the antibody in the aqueous deliverysystem.

The compositions may be administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound (e.g., CD40-specific antibody)employed; the metabolic stability and length of action of the compound;the age, body weight, general health, sex, and diet of the patient; themode and time of administration; the rate of excretion; the drugcombination; the severity of the particular disorder or condition; andthe subject undergoing therapy. Generally, a therapeutically effectivedaily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeuticallyeffective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeuticallyeffective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg)to about 25 mg/kg (i.e., 1.75 g).

Compositions comprising the CD40-specific antibodies of the presentdisclosure may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy may include administration of a singlepharmaceutical dosage formulation which contains a compound of theinvention and one or more additional active agents, as well asadministration of compositions comprising antibodies of the inventionand each active agent in its own separate pharmaceutical dosageformulation. For example, an antibody as described herein and the otheractive agent can be administered to the patient together in a singleoral dosage composition such as a tablet or capsule, or each agentadministered in separate oral dosage formulations. Similarly, anantibody as described herein and the other active agent can beadministered to the patient together in a single parenteral dosagecomposition such as in a saline solution or other physiologicallyacceptable solution, or each agent administered in separate parenteraldosage formulations. Where separate dosage formulations are used, thecompositions comprising antibodies and one or more additional activeagents can be administered at essentially the same time, i.e.,concurrently, or at separately staggered times, i.e., sequentially andin any order; combination therapy is understood to include all theseregimens.

Thus, in certain embodiments, also contemplated is the administration ofanti-CD40 antibody compositions of this disclosure in combination withone or more other therapeutic agents. Such therapeutic agents may beaccepted in the art as a standard treatment for a particular diseasestate as described herein, such as rheumatoid arthritis, inflammation orcancer.

Exemplary therapeutic agents contemplated include cytokines, growthfactors, steroids, NSAIDs, DMARDs, anti-inflammatories,chemotherapeutics, radiotherapeutics, or other active and ancillaryagents.

In certain embodiments, the anti-CD40 antibodies disclosed herein may beadministered in conjunction with any number of chemotherapeutic agents.Examples of chemotherapeutic agents include alkylating agents such asthiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A variety of other therapeutic agents may be used in conjunction withthe anti-CD40 antibodies described herein. In one embodiment, theantibody is administered with an anti-inflammatory agent.Anti-inflammatory agents or drugs include, but are not limited to,steroids and glucocorticoids (including betamethasone, budesonide,dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone),nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin,ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNFmedications, cyclophosphamide and mycophenolate.

Exemplary NSAIDs are chosen from the group consisting of ibuprofen,naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib)and CELEBREX® (celecoxib), and sialylates. Exemplary analgesics arechosen from the group consisting of acetaminophen, oxycodone, tramadolof proporxyphene hydrochloride. Exemplary glucocorticoids are chosenfrom the group consisting of cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, or prednisone. Exemplary biologicalresponse modifiers include molecules directed against cell surfacemarkers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNFantagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) andinfliximab (REMICADE®)), chemokine inhibitors and adhesion moleculeinhibitors. The biological response modifiers include monoclonalantibodies as well as recombinant forms of molecules. Exemplary DMARDsinclude azathioprine, cyclophosphamide, cyclosporine, methotrexate,penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold(oral (auranofin) and intramuscular) and minocycline.

In certain embodiments, the antibodies described herein are administeredin conjunction with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormones such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and —II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

The compositions comprising herein described CD40-specific antibodiesmay be administered to an individual afflicted with a disease asdescribed herein, including, but not limited to non-Hodgkin's lymphomas,Hodgkin's lymphoma, chronic lymphocytic leukemias, hairy cell leukemias,acute lymphoblastic leukemias, multiple myeloma, carcinomas of thepancreas, colon, gastric intestine, prostate, bladder, kidney ovary,cervix, breast, lung, nasopharynx, malignant melanoma and rituximabresistant NHL and leukemias, autoimmune and inflammatory diseases.Autoimmune diseases include but are not limited to, arthritis (includingrheumatoid arthritis, reactive arthritis), systemic lupus erythematosus(SLE), psoriasis and inflammatory bowel disease (IBD),encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis,insulin dependent diabetes, Addison's disease, celiac disease, chronicfatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosingspondylitis, ulcerative colitis, Crohn's disease, fibromyalgia,pemphigus vulgaris, Sjogren's syndrome, Kawasaki's Disease,hyperthyroidism/Graves disease, hypothyroidism/Hashimoto's disease,endometriosis, scleroderma, pernicious anemia, Goodpasture syndrome,Guillain-Barré syndrome, Wegener's disease, glomerulonephritis, aplasticanemia (including multiply transfused aplastic anemia patients),paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome,idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evan'ssyndrome, Factor VIII inhibitor syndrome, systemic vasculitis,dermatomyositis, polymyositis and rheumatic fever, autoimmunelymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid,Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis,and autoimmune myocarditis.

Inflammatory diseases include, but are not limited to, Crohn's disease,colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritablebowel syndrom (IBS), lupus erythematous, nephritis, Parkinson's disease,ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease,arthritis, rheumatoid arthritis, asthma, and various cardiovasculardiseases such as atherosclerosis and vasculitis. In certain embodiments,the inflammatory disease is selected from the group consisting ofrheumatoid arthritis, diabetes, gout, cryopyrin-associated periodicsyndrome, and chronic obstructive pulmonary disorder. In this regard,one embodiment provides a method of treating, reducing the severity ofor preventing inflammation or an inflammatory disease by administeringto a patient in need thereof a therapeutically effective amount of aherein disclosed compositions comprising anti-CD40 antibodies.

For in vivo use for the treatment of human disease, the antibodiesdescribed herein are generally incorporated into a pharmaceuticalcomposition prior to administration. A pharmaceutical compositioncomprises one or more of the antibodies described herein in combinationwith a physiologically acceptable carrier or excipient as describedelsewhere herein. To prepare a pharmaceutical composition, an effectiveamount of one or more of the compounds is mixed with any pharmaceuticalcarrier(s) or excipient known to those skilled in the art to be suitablefor the particular mode of administration. A pharmaceutical carrier maybe liquid, semi-liquid or solid. Solutions or suspensions used forparenteral, intradermal, subcutaneous or topical application mayinclude, for example, a sterile diluent (such as water), salinesolution, fixed oil, polyethylene glycol, glycerin, propylene glycol orother synthetic solvent; antimicrobial agents (such as benzyl alcoholand methyl parabens); antioxidants (such as ascorbic acid and sodiumbisulfite) and chelating agents (such as ethylenediaminetetraacetic acid(EDTA)); buffers (such as acetates, citrates and phosphates). Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, polypropylene glycol and mixtures thereof.

The compositions comprising CD40-specific antibodies as described hereinmay be prepared with carriers that protect the antibody against rapidelimination from the body, such as time release formulations orcoatings. Such carriers include controlled release formulations, suchas, but not limited to, implants and microencapsulated delivery systems,and biodegradable, biocompatible polymers, such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, PEGs, polyorthoesters,polylactic acid and others known to those of ordinary skill in the art.

Provided herein are methods of treatment using the antibodies that bindCD40. In one embodiment, an antibody of the present invention isadministered to a patient having a disease involving inappropriateexpression of CD40, which is meant in the context of the presentdisclosure to include diseases and disorders characterized by aberrantCD40 expression or activity, due for example to alterations (e.g.,statistically significant increases or decreases) in the amount of aprotein present, or the presence of a mutant protein, or both. Anoverabundance may be due to any cause, including but not limited tooverexpression at the molecular level, prolonged or accumulatedappearance at the site of action, or increased (e.g., in a statisticallysignificant manner) activity of CD40 relative to that which is normallydetectable. Such an overabundance of CD40 can be measured relative tonormal expression, appearance, or activity of CD40 signalling events,and said measurement may play an important role in the developmentand/or clinical testing of the antibodies described herein.

The present antibodies are useful for the treatment of a variety ofcancers. In certain embodiments, the antibodies described herein exertanti-tumor activity by activating anti-tumor immune responses. Incertain embodiments, the present antibodies are useful for the treatmentof a variety of cancers associated with the aberrant expression of CD40.In one embodiment of the invention provides a method for the treatmentof a cancer including, but not limited to, non-Hodgkin's lymphomas,Hodgkin's lymphoma, chronic lymphocytic leukemias, hairy cell leukemias,acute lymphoblastic leukemias, multiple myeloma, carcinomas of thepancreas, colon, gastric intestine, prostate, bladder, kidney ovary,cervix, breast, lung, nasopharynx, malignant melanoma and rituximabresistant NHL and leukemias, by administering to a cancer patient atherapeutically effective amount of a herein disclosed CD40-specificantibody. An amount that, following administration, inhibits, preventsor delays the progression and/or metastasis of a cancer in astatistically significant manner (i.e., relative to an appropriatecontrol as will be known to those skilled in the art) is consideredeffective.

Another embodiment provides a method for preventing metastasis of acancer including, but not limited to, non-Hodgkin's lymphomas, Hodgkin'slymphoma, chronic lymphocytic leukemias, hairy cell leukemias, acutelymphoblastic leukemias, multiple myeloma, carcinomas of the pancreas,colon, gastric intestine, prostate, bladder, kidney ovary, cervix,breast, lung, nasopharynx, malignant melanoma and rituximab resistantNHL and leukemias, by administering to a cancer patient atherapeutically effective amount of a herein disclosed CD40-specificantibody (e.g., an amount that, following administration, inhibits,prevents or delays metastasis of a cancer in a statistically significantmanner, i.e., relative to an appropriate control as will be known tothose skilled in the art).

Another embodiment provides a method for preventing a cancer including,but not limited to, non-Hodgkin's lymphomas, Hodgkin's lymphoma, chroniclymphocytic leukemias, hairy cell leukemias, acute lymphoblasticleukemias, multiple myeloma, carcinomas of the pancreas, colon, gastricintestine, prostate, bladder, kidney ovary, cervix, breast, lung,nasopharynx, malignant melanoma and rituximab resistant NHL andleukemias, by administering to a cancer patient a therapeuticallyeffective amount of a herein disclosed CD40-specific antibody.

Another embodiment provides a method for treating, ameliorating thesymptoms of, inhibiting the progression of or prevention ofnon-Hodgkin's lymphomas, Hodgkin's lymphoma, chronic lymphocyticleukemias, hairy cell leukemias, acute lymphoblastic leukemias, multiplemyeloma, carcinomas of the pancreas, colon, gastric intestine, prostate,bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignantmelanoma and rituximab resistant NHL and leukemias by administering to apatient afflicted by one or more of these diseases a therapeuticallyeffective amount of a herein disclosed CD40-specific antibody.

Another embodiment provides a method for treating, ameliorating thesymptoms of, inhibiting the progression of or prevention of anautoimmune disease by administering to a patient afflicted by one ormore of these diseases a therapeutically effective amount of a hereindisclosed anti-CD40 antibody. In this regard, autoimmune diseasesinclude, but are not limited to, arthritis (including rheumatoidarthritis, reactive arthritis), systemic lupus erythematosus (SLE),psoriasis and inflammatory bowel disease (IBD), encephalomyelitis,uveitis, myasthenia gravis, multiple sclerosis, insulin dependentdiabetes, Addison's disease, celiac disease, chronic fatigue syndrome,autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis,ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris,Sjogren's syndrome, Kawasaki's Disease, hyperthyroidism/Graves disease,hypothyroidism/Hashimoto's disease, endometriosis, scleroderma,pernicious anemia, Goodpasture syndrome, Guillain-Barr{tilde over (e)}syndrome, Wegener's disease, glomerulonephritis, aplastic anemia(including multiply transfused aplastic anemia patients), paroxysmalnocturnal hemoglobinuria, myelodysplastic syndrome, idiopathicthrombocytopenic purpura, autoimmune hemolytic anemia, Evan's syndrome,Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis,polymyositis and rheumatic fever, autoimmune lymphoproliferativesyndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease,sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmunemyocarditis.

Another embodiment provides a method for treating, ameliorating thesymptoms of, inhibiting the progression of or prevention of aninflammatory disease by administering to a patient afflicted by one ormore of these diseases a therapeutically effective amount of a hereindisclosed anti-CD40 antibody.

Inflammatory diseases include, but are not limited to, Crohn's disease,colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritablebowel syndrom (IBS), lupus erythematous, nephritis, Parkinson's disease,ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease,arthritis, rheumatoid arthritis, asthma, and various cardiovasculardiseases such as atherosclerosis and vasculitis. In certain embodiments,the inflammatory disease is selected from the group consisting ofrheumatoid arthritis, diabetes, gout, cryopyrin-associated periodicsyndrome, and chronic obstructive pulmonary disorder.

In another embodiment, anti-CD40 antibodies of the present invention areused to determine the structure of bound antigen, e.g., conformationalepitopes, which structure may then be used to develop compounds havingor mimicking this structure, e.g., through chemical modeling and SARmethods.

Various other embodiments of the present invention relate, in part, todiagnostic applications for detecting the presence of cells or tissuesexpressing CD40. Thus, the present disclosure provides methods ofdetecting CD40 in a sample, such as detection of cells or tissuesexpressing CD40. Such methods can be applied in a variety of knowndetection formats, including, but not limited to immunohistochemistry(IHC), immunocytochemistry (ICC), in situ hybridization (ISH),whole-mount in situ hybridization (WISH), fluorescent DNA in situhybridization (FISH), flow cytometry, enzyme immuno-assay (EIA), andenzyme linked immuno-assay (ELISA).

ISH is a type of hybridization that uses a labeled complementary DNA orRNA strand (i.e., primary binding agent) to localize a specific DNA orRNA sequence in a portion or section of a cell or tissue (in situ), orif the tissue is small enough, the entire tissue (whole mount ISH). Onehaving ordinary skill in the art would appreciate that this is distinctfrom immunohistochemistry, which localizes proteins in tissue sectionsusing an antibody as a primary binding agent. DNA ISH can be used ongenomic DNA to determine the structure of chromosomes. Fluorescent DNAISH (FISH) can, for example, be used in medical diagnostics to assesschromosomal integrity. RNA ISH (hybridization histochemistry) is used tomeasure and localize mRNAs and other transcripts within tissue sectionsor whole mounts.

In various embodiments, the antibodies described herein are conjugatedto a detectable label that may be detected directly or indirectly. Inthis regard, an antibody “conjugate” refers to an anti-CD40 antibodythat is covalently linked to a detectable label. In the presentinvention, DNA probes, RNA probes, monoclonal antibodies,antigen-binding fragments thereof, and antibody derivatives thereof,such as a single-chain-variable-fragment antibody or an epitope taggedantibody, may all be covalently linked to a detectable label. In “directdetection”, only one detectable antibody is used, i.e., a primarydetectable antibody. Thus, direct detection means that the antibody thatis conjugated to a detectable label may be detected, per se, without theneed for the addition of a second antibody (secondary antibody).

A “detectable label” is a molecule or material that can produce adetectable (such as visually, electronically or otherwise) signal thatindicates the presence and/or concentration of the label in a sample.When conjugated to a antibody, the detectable label can be used tolocate and/or quantify the target to which the specific antibody isdirected. Thereby, the presence and/or concentration of the target in asample can be detected by detecting the signal produced by thedetectable label. A detectable label can be detected directly orindirectly, and several different detectable labels conjugated todifferent specific-antibodies can be used in combination to detect oneor more targets.

Examples of detectable labels, which may be detected directly, includefluorescent dyes and radioactive substances and metal particles. Incontrast, indirect detection requires the application of one or moreadditional antibodies, i.e., secondary antibodies, after application ofthe primary antibody. Thus, the detection is performed by the detectionof the binding of the secondary antibody or binding agent to the primarydetectable antibody. Examples of primary detectable binding agents orantibodies requiring addition of a secondary binding agent or antibodyinclude enzymatic detectable binding agents and hapten detectablebinding agents or antibodies.

In some embodiments, the detectable label is conjugated to a nucleicacid polymer which comprises the first binding agent (e.g., in an ISH,WISH, or FISH process). In other embodiments, the detectable label isconjugated to an antibody which comprises the first binding agent (e.g.,in an IHC process).

Examples of detectable labels which may be conjugated to antibodies usedin the methods of the present disclosure include fluorescent labels,enzyme labels, radioisotopes, chemiluminescent labels,electrochemiluminescent labels, bioluminescent labels, polymers, polymerparticles, metal particles, haptens, and dyes.

Examples of fluorescent labels include 5-(and 6)-carboxyfluorescein, 5-or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoicacid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, anddyes such as Cy2, Cy3, and Cy5, optionally substituted coumarinincluding AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE)and allophycoerythrin (APC), Texas Red, Princeton Red, green fluorescentprotein (GFP) and analogues thereof, and conjugates of R-phycoerythrinor allophycoerythrin, inorganic fluorescent labels such as particlesbased on semiconductor material like coated CdSe nanocrystallites.

Examples of polymer particle labels include micro particles or latexparticles of polystyrene, PMMA or silica, which can be embedded withfluorescent dyes, or polymer micelles or capsules which contain dyes,enzymes or substrates.

Examples of metal particle labels include gold particles and coated goldparticles, which can be converted by silver stains. Examples of haptensinclude DNP, fluorescein isothiocyanate (FITC), biotin, and digoxigenin.Examples of enzymatic labels include horseradish peroxidase (HRP),alkaline phosphatase (ALP or AP), β-galactosidase (GAL),glucose-6-phosphate dehydrogenase, β-N-acetylglucosamimidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase andglucose oxidase (GO). Examples of commonly used substrates forhorseradishperoxidase include 3,3′-diaminobenzidine (DAB),diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole(AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR),Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol(CN), .alpha.-naphtol pyronin (.alpha.-NP), o-dianisidine (OD),5-bromo-4-chloro-3-indolylphosp-hate (BCIP), Nitro blue tetrazolium(NBT), 2-(p-iodophenyl)-3-p-nitropheny-I-5-phenyl tetrazolium chloride(INT), tetranitro blue tetrazolium (TNBT),5-bromo-4-chloro-3-indoxyl-beta-D-galactoside/ferro-ferricyanide(BCIG/FF).

Examples of commonly used substrates for Alkaline Phosphatase includeNaphthol-AS-B1-phosphate/fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/-fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/new fuschin (NABP/NF), bromochloroindolylphosphate/nitroblue tetrazolium (BCIP/NBT),5-Bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

Examples of luminescent labels include luminol, isoluminol, acridiniumesters, 1,2-dioxetanes and pyridopyridazines. Examples ofelectrochemiluminescent labels include ruthenium derivatives. Examplesof radioactive labels include radioactive isotopes of iodide, cobalt,selenium, tritium, carbon, sulfur and phosphorous.

Detectable labels may be linked to the antibodies described herein or toany other molecule that specifically binds to a biological marker ofinterest, e.g., an antibody, a nucleic acid probe, or a polymer.Furthermore, one of ordinary skill in the art would appreciate thatdetectable labels can also be conjugated to second, and/or third, and/orfourth, and/or fifth binding agents or antibodies, etc. Moreover, theskilled artisan would appreciate that each additional binding agent orantibody used to characterize a biological marker of interest may serveas a signal amplification step. The biological marker may be detectedvisually using, e.g., light microscopy, fluorescent microscopy, electronmicroscopy where the detectable substance is for example a dye, acolloidal gold particle, a luminescent reagent. Visually detectablesubstances bound to a biological marker may also be detected using aspectrophotometer. Where the detectable substance is a radioactiveisotope detection can be visually by autoradiography, or non-visuallyusing a scintillation counter. See, e.g., Larsson, 1988,Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.);Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (HumanaPress, Totowa, N.J.).

The invention further provides kits for detecting CD40 or cells ortissues expressing CD40 in a sample, wherein the kits contain at leastone antibody, polypeptide, polynucleotide, vector or host cell asdescribed herein. In certain embodiments, a kit may comprise buffers,enzymes, labels, substrates, beads or other surfaces to which theantibodies of the invention are attached, and the like, and instructionsfor use.

EXAMPLES Example 1 Production and Humanization of Anti-CD40 Antibodies

Four New Zealand white rabbits were immunized with recombinant rabbitFc-hCD40. The rabbit with the highest serum titers of specific bindingto human CD40 was chosen for cell fusion. A total of 172 hybridomas wereidentified as positive binders to soluble Fc-hCD40, of which 44 cloneswere found to be positive binders to cell surface CD40. After theepitope clustering assay, 24 representative hybridomas were selected forrecombinant expression and further characterization. Secondaryfunctional screening was carried out as described further below andincluded: 1) induction of DC maturation as measured by CD80, CD83, CD86upregulation (agonist activity); 2) induction of direct tumor growthinhibition (agonist activity); and 3) ADCC antibody effector function.Candidates were selected based on dual functional screenings whichincluded two arms: 1) binding affinity, antibody internalization,antibody dependent cellular cytotoxicity (ADCC), complement dependentcytotoxicty (CDC), and antibody dependent cellular phagocytosis (ADCP);and 2) agonist DC activation/maturation function, receptor-ligandinteraction, mixed lymphocyte reaction (MLR), cell proliferation andapoptosis.

Screening Agonist Antibodies Via Dendritic Cell Maturation

To further clarify the agonist or antagonist effect of the initial panelof anti-CD40 antibodies, a DC maturation assay was used as an indicatorto screen for functional antibodies. Anti-CD40 or control antibodieswere added to human monocyte-derived DC culture solution for 2 days.Upregulation of CD83, one of the best-known maturation markers for humandendritic cells, was measured to screen for agonist antibodies. 5C11, amouse monoclonal antibody that induces dendritic cell maturation wasused as positive control. Antibodies R-3, R-6, R-8, R-9, R-16, R-18,R-24, R-33, R-36, 19-21, 19-45 and 19-59 increased more than 50% of CD83expression as compared with Ig control (FIG. 1A). DC maturation wasfurther determined by measuring the antibody-induced up-regulation ofco-stimulatory molecules CD80 and CD86 for the selected antibodies. Asshown in FIG. 1B and FIG. 1C, antibodies R-3, R-8, R-9, R-33 and 19-21up-regulated both CD80 and CD86, while the other antibodies had onlymodest effects. These results were consistent with the CD83 modulationeffects by these antibodies. Interestingly, among the antibodies capableof inducting DC maturation, only clone 19-21 showed strong activity toenhance T cells proliferation in a mixed-lymphocyte reaction (FIG. 1D).

Screening for Direct Inhibition of Tumor Growth

The panel of agonist anti-CD40 antibodies was further assessed for theability to induce the tumor growth inhibition of CD40 expressing tumorcells. All anti-CD40 antibodies tested inhibited tumor cellproliferation. The antibody 19-21 demonstrated the highest potency.(FIG. 2).

Screening for ADCC Activity

In addition to induction of APC activation and tumor growth inhibition,antibody effector function, ADCC, was used as an important criterion toscreen and rank the antibody candidates. In order to conduct the ADCCassay using human PBMC, all the selected antibodies were converted fromrabbit mAb to chimeric mAb with rabbit Fab and human IgG1. As shown inFIG. 3, all the selected candidates showed significant ADCC activity ascompared to IgG1 control. Based on the maximal ADCC activity, the leadmAbs can be ranked cR-8>cR-3>cR-33>c19-21>c R-9>c19-59.

Four candidates (c19-21, cR-8, cR-3, cR-33) were selected based on invitro functional screening. Their in vitro characterizations aresummarized in Table 1. Antibody c19-21strongly enhances DC activationand tumor growth inhibition, while antibodies cR-8 and cR-3 showed morepotent ADCC activity.

TABLE 1 Characteristics of 4 candidate anti-CD40 antibodies: Antibodyc19-21 cR-8 cR3 cR33 Blocks CD40L binding Yes Yes Yes Yes Enhances DCmaturation Strong Strong Strong Strong Pro- apoptosis activity 0.02μg/ml 0.45 μg/ml 0.57 μg/ml 0.90 μg/ml (IC50) ADCC (% cytotoxicity 26%33% 30% 29% of Ramos cell at 1 μg/ml)

In Vivo Anti-Tumor Activity Screening

As the top 4 candidates showed different potencies in different in vitroassays, we conducted in vivo studies to evaluate and compare theiranti-tumor activity for lead selection. The Ramos tumor xenograft modelwas used. Tumor bearing mice were treated i.p. with 5 mg/kg of chimericantibodies cR-3, cR-8, cR-33 or c19-21 3 times per week for a total of 9doses (eight animals per group). The anti-tumor activity of rituximabwith the same regimen was used as a reference. As shown in FIG. 4A, cR-8and cR-3 showed the strongest anti-tumor effect. In contrast, 19-21exhibited lower anti-tumor activity with faster tumor rebound aftertermination of dosing. The anti-tumor effect of cR-33 was in between,but still exhibited better in vivo efficacy than rituximab. The in vivopotency of antibodies cR-3 and cR-8 was further evaluated in adose-response study. As shown in FIG. 4B, cR-8 showed more potentanti-tumor efficacy than cR-3, and thus was identified as the leadanti-CD40 antibody.

The amino acid sequence of the heavy chain and light chain variableregions of the R-8 clone are set forth in SEQ ID NOs:1 and 2. The aminoacid sequences of the CDRs of the VH and VL are set forth in SEQ IDNOs:3-5 and 6-8, respectively. The amino acid sequence of the heavy andlight chain sequences of several of the other antibody candidates thatshowed functional activity are set forth in SEQ ID NOs:11-56. VHCDR andVLCDR amino acid sequences for these antibodies are provided in SEQ IDNos:57-194. FIG. 16 shows an alignment of these sequences, including theR-8 clone, with CDRs underlined.

R-8 was humanized using a proprietary mutational lineage guided (MLG)humanization technology (see e.g., U.S. Pat. No. 7,462,697). The lightand heavy chain framework of the humanized R-8 (APX005) are 95%identical to the human germline sequences. The amino acid sequence ofthe humanized VH and VL regions are set forth in SEQ ID NOs:9 and 10,respectively. The binding of APX005 to CD40 were found to be similar toits parental clone R-8.

Example 2 In Vitro Characterization of the APX005 Humanized Anti-CD40Antibody

Numerous in vitro experiments were conducted to further characterize theAPX005 humanized antibody.

APX005 Selectively Binds to CD40

Binding selectivity of APX005 was assessed by direct ELISA to a panel ofTNFR family proteins. A total of 1 μg/ml of fusion protein of rabbit Fcand CD40, RANK, TweakR, OX40, DR5 and 4-1BB were coated on ELISA plates.Bound APX005 was detected using goat anti-human HRP-conjugated IgG. Asshown in FIG. 5, APX005 selectively binds to human CD40 but not otherTNFR family proteins tested.

APX005 Blocks Binding of CD40L to CD40

An ELISA was conducted to assess the effect of APX005 on CD40L bindingto CD40. In particular, CD40L (4 μg/ml final concentration) was used tobind the immobilized human CD40 onto an ELISA plate, and changes in thebinding amount of CD40L to CD40 were measured after pre-incubatingimmobilized CD40 with APX005. CD40L binding to immobilized CD40 wasdetected by a mouse anti-CD40L monoclonal antibody. As shown in FIG. 6,APX005 blocks the binding of CD40L to CD40. In contrast, SGN-40increases the binding.

APX005/CD40 Complex is not Internalized

In order to assess the target mediated-internalization of APX005 forevaluating its impact on ADCC activity, Ramos cells were incubated withAPX005 for 4 h at 37° C., a temperature permissive for internalization,or at 4° C. for 30 minutes, a temperature at which internalization isminimized. Cells were washed with staining buffer, followed byincubation with Alexa 488 labeled goat anti-human IgG for an additional30 minutes at 4° C. FACS analysis was performed to examine the level ofAPX005 on the cell surface. As shown in FIG. 7, there was no reduction(slight increase) of APX005 level on the cell surface after incubationat 37° C. The data suggest that upon binding to CD40 APX005/CD40 complexwas not internalized by tumor cells, thus providing optimal conditionsfor recruiting the effector cells for ADCC.

APX005 Mediates ADCC

In order to assess ADCC activity of APX005 on CD40 expressing tumorcells, CD40 expressing Ramos and Daudi were used as target cells andfresh human peripheral blood mononuclear cell (PBMC) were used as theeffector cells. ADCC was measured by a calcein-AM release assay. Targetcells were labeled with calcein-AM (15 uM/10⁶ cells), washed, and platedin triplicate at 5×10³ per well in round-bottomed 96-well plates.Increasing concentrations (0.0001-10 μg/mL) of either APX005 or controlantibodies were pre-incubated at 4° C. for 30 minutes, after which PBMCeffector cells from healthy human donors were added with a finaleffector:target cell ratio of 40:1 in a final volume of 200 uL per well.Experiments were performed using PBMC from at least three differentdonors. After a 4-hour incubation, 100 μL culture supernatants weretransferred to a Black View Plate-96 plate and arbitrary fluorescentunits (AFU) were read on a Victor II plate reader (485 nm excitation/535nm emission). Percent specific lysis=(AFU mean experimental release—AFUmean spontaneous release)/(AFU mean maximal release—AFU mean spontaneousrelease). As showing in FIG. 8, APX005 induced ADCC in a dose-dependentfashion. A similar effect was observed for SGN-40. The differentsensitivity of Ramos and Daudi cells to the ADCC may be due to differentCD40 expression level (Cancer Res 2005; 65: 8331-8338).

APX005 Inhibits Tumor Cell Proliferation

To assess the ability of APX005 to inhibit tumor cell proliferation,Ramos cells were seeded in 96-well flat-bottom plates at 50,000cells/well in 200 μL RPMI 1640 supplemented with 10% FBS containingvarying concentrations of APX005, SGN-40 or a control human IgG. Forcross-linking, APX005, SGN-40 or control IgG was pre-inbubated withF(ab′)2 fragments of a goat anti-human IgG Fc fragment-specific antibodyin the medium for 30 minutes at room temperature before being added tothe cells. Cells were treated for a total of 72 hours. Then 10%AlamarBlue® (Serotec, Oxford, UK) was added to each well and incubatedfor an additional 24 hours. Cell viability was measured by a CytoFluorfluorescence reader with an excitation wavelength of 530 nm and emissionwavelength of 590 nm. All studies were conducted twice and intriplicates for each sample concentration. As shown in FIG. 9, monomerAPX005 inhibited proliferation of Ramos cells (FIG. 9A). When APX005 wascross-linked by a secondary antibody, it delivered an increased anddose-dependent proliferation inhibitory effect (FIG. 9B). Thecross-linking of APX005 can be achieved in vivo by Fc receptorexpressing cells.

APX005 Induces DC Activation

In order to assess the ability of APX005 to stimulate the maturation ofDC cells, PBMC were prepared by density gradient centrifugation usinglymphocyte isolation solution. Adherent monocytes were harvested afterincubating for 2 hours at 37° C. Isolated monocytes were cultured with100 ng/ml of recombinant human GM-CSF and 100 ng/ml of recombinant humanIL-4 in RPM11640 media supplemented with 10% FCS in a 24-well plate.Half of the medium was changed after 3 days. On day 5 of culturing, 1.3nM of anti-CD40 antibodies, CD40L or the control antibody were added tothe DC cells, and further cultured for 48 h in a 24-well plate. For DCactivation marker staining, PE-conjugated anti-CD83, anti-CD86 antibodyand anti-CD80 antibody were used. Analysis was performed using FACS. Thedata is from one representative study. As shown in FIG. 10, APX005induced marked DC maturation and its effect appears more potent thanSGN-40 and CD40L. Increased activation of DC may lead to more potentanti-tumor T cell responses.

APX005 is Cross Reactive with Monkey CD40 but not Mouse CD40

Cross-reactivity was assessed by direct ELISA. A total of 1 μg/ml ofhuman CD40, monkey CD40 or mouse CD40 was coated on ELISA platesfollowed by incubation with 1 μg/ml of APX005 or control IgG1.Antibodies bound to CD40 were detected using goat anti-human IgGconjugated with HRP. APX005 clearly crossreacts with monkey CD40 but notmouse CD40. (FIG. 11A) Cross-reactivity of APX005 with mouse CD40 wasfurther determined by FACS binding to a mouse A20 cell line whichexpresses mouse CD40. An aliquot of 0.5×10⁶ A20 cells was added to 96well plates and incubated with 100 μl diluted rat anti-mouse CD40antibody conjugated with PE, APX005 or IgG1 control antibodies. Afterwashing, 100 μl Goat-anti human IgG (H+L) conjugated with R-PE (SouthernBiotech CAT#2040-09) were added at 1:200 dilution in PBS to the sampleand incubated for detection of APX005 and control human IgG1. A ratanti-mouse CD40 antibody conjugated with PE was used as positivecontrol. Samples were re-suspended with 0.5 ml PBS and analyzed by FACS.The FACS data showed that APX005 does not crossreact with mouse CD40(FIG. 11B).

In summary, the experiments in this Example showed that APX005 is ahumanized IgG1 antibody that binds CD40. APX005 specifically binds toCD40 with a K_(d) of 9.6×10⁻¹⁰ M and blocks CD40L binding to CD40. Thisis in contrast to the SGN40 anti-CD40 antibody that enhances theCD40-CD40L interaction. This suggests that these two antibodies bind todistinct epitopes. In vitro, APX005 showed potent ADCC activity to CD40positive lymphoma cells (Ramos and Daudi) as well as the ability todirectly inhibit tumor cell (Ramos) proliferation upon cross-linking.APX005 also stimulated the maturation of dendritic cells to enhancecellular immune response. Additionally, APX005 was shown to cross-reactwith monkey CD40.

Example 3 In Vivo Characterization of the APX005 Humanized Anti-CD40Antibody

Numerous in vivo experiments were conducted to further characterize theAPX005 humanized antibody.

APX005 Inhibition of Tumor Growth in the Ramos Model

In order to evaluate the effect of APX005 on the xenograft model ofhuman B cell lymphoma, female BALB/c nu/nu mice 6-8 weeks of age wereused for tumor cell innoculation. Xenografts were established bysubcutaneous inoculation of 1×10⁷ tumor cells/mouse into the dorsalflanks. When tumors reached an average volume of about 100 mm3 (50-200mm3), the animals were randomized into groups. Antibodies wereadministered intraperitoneally at 3 mg/kg starting at day 13 (see FIG.12). Dosing was administered 3 times per week for a total of 9 doses(eight animals per group). Perpendicular dimensions of the tumor weremeasured using a Vernier scale caliper. Tumor volumes were calculatedusing the formula: Volume=(length×width²)/2. As shown in FIG. 12A,APX005 demonstrated potent and long-lasting anti-tumor activity. Serumwas taken at day 34, two days after the last dosing, for determining invivo drug levels by measuring human IgG concentrations (see FIG. 12B).The anti-tumor efficacy mediated by APX005 was greater than that ofSGN-40 and persisted long after the dosing period. Single point PKanalysis showed that the superior anti-tumor activity of APX005 was notdue to PK difference.

APX005 Inhibition of Rituximab Pre-Treated and Resistant Tumors

The purpose of this experiment was to evaluate the effect of APX005 onrituximab pre-treated and resistant B cell lymphoma. Nude mice bearingestablished Ramos tumors were first treated with rituximab at 3 mg/kgfor 5 doses. Tumor growth was partially inhibited by rituximab (FIG.13A). When these tumors reached size about 700 mm³, they were randomizedinto 4 groups (7 animals per group) and re-treated i.p. with APX005,rituximab, SGN40 analog 3 mg/kg or saline control for 3 weeks (FIG.13B). As shown in FIG. 13, rituximab pre-treated tumors failed torespond to rituximab re-treatment, suggesting that these tumors arerituximab resistant (FIG. 13B). APX005 exhibited the capability ofinhibiting the growth of rituximab resistant tumors.

APX005 Inhibition of Tumor Growth in the Raji Model

The purpose of this experiment was to determine the dose and efficacyrelationship of APX005 in vivo. Nude mice bearing established CD40positive Raji tumors were treated with APX005 starting at day 15. Dosesof APX005 ranging from 0.1 mg/kg-10 mg/kg were administered i.p. 3times/week for 2 weeks (eight animals per group) (see FIG. 14). Salinewas used as control treatment. Tumor volumes were measured on eachdosing day. Serum levels of APX005 in each group were also measured 3days after last dosing to determine the correlation of the in vivoefficacy with the levels of APX005 in the circulation. Cleardose-dependent anti-tumor activity was observed (see FIG. 14).Differences in tumor volumes were significant (P<0.05) between thecontrol group and antibody treatment groups with dose levels ≥1 mg/kg ondays 29 to 33. The minimal effective dose was determined as 1 mg/kg,which corresponded to a median serum concentration of 0.49 μg/ml at day36. Differences in tumor volumes between the 3, 5 and 10 mg/kg dosegroups were not statistically significant. Thus, the maximal anti-tumoractivity was achieved at doses >3 mg/kg with a median serumconcentration ≥1.6 μg/ml.

Inhibition of Tumor Growth in Human MM IM-9 Model by APX005

In order to evaluate the anti-tumor activity of APX005 in human multiplemyeloma model, nude mice bearing established CD40 positive multiplemyeloma IM-9 tumors were treated i.p. with APX005 or SGN40 starting atday 15. APX005 was given at 3 mg/kg, 3 times/week for 3 weeks (5 animalsper group). Tumor volumes were measured on each dosing day.

APX005 demonstrated potent anti-tumor activity against human multiplemyeloma in the IM-9 xenograft model (see FIG. 15). The anti-tumorefficacy mediated by APX005 was significantly greater than that ofSGN-40 (P<0.05).

Inhibition of Tumor Growth in the Ramos Model by APX005 as Compared withSGN-40 and Rituximab

The purpose of this experiment was to compare the anti-tumor activity ofAPX005, rituximab and SGN-40 in a human B cell lymphoma Ramos xenograftmodel. Xenografts were established by subcutaneous inoculation of Ramoscells into the dorsal flanks of female SCID C.B-17 mice. When tumorsreached an average volume of about 200-300 mm³, the animals wererandomized into 6 groups. Antibodies were administered intraperitoneallyat doses as indicated in FIG. 17. Dosing was administered 3 times perweek for a total of 9 doses (10 animals per group). Perpendiculardimensions of the tumor were measured using a Vernier scale caliper.Tumor volumes were calculated using the formula:Volume=(length×width²)/2. Survival of the mice was also determined andrecorded.

APX005 demonstrated dose-dependent anti-tumor activity. Treatment withthe high dose of APX005 (10 mg/kg) resulted in complete tumorregression, while rituximab at the same dose (10 mg/kg) only delayedtumor growth, suggesting that APX005 is more efficacious than rituximabin this model. APX005 is also more potent than SGN-40 (FIG. 17A). APX005not only inhibited tumor growth but also improved the survival of thetumor bearing animals (FIG. 17B).

Inhibition of Tumor Growth in a Rituximab-Resistant Human NamalwaLymphoma Xenograft Model

The purpose of this experiment was to compare the anti-tumor activity ofAPX005, rituximab and SGN-40 in the rituximab-resistant human Namalwalymphoma model. Xenografts were established by subcutaneous inoculationof Namalwa cells into the dorsal flanks of female SCID C.B-17 mice. Whentumors reached an average volume of about 200-300 mm3, the animals wererandomized into 6 groups. Antibodies were administered i.p. at the dosesindicated in FIG. 18. Dosing was administered 3 times per week for atotal of 9 doses (10 animals per group). Perpendicular dimensions of thetumor were measured using a Vernier scale caliper. Tumor volumes werecalculated using the formula: Volume=(length×width²)/2. Survival of themice was also determined and recorded.

APX005 demonstrated potent anti-tumor activity in rituximab-resistantNamalwa lymphoma model (FIG. 18A). APX005 also improved the survival ofmice bearing rituximab-resistant tumors (FIG. 18B).

In summary, the experiments in this Example showed that the efficacy ofAPX005 was examined in multiple xenograft tumor models. APX005 markedlyinhibited the tumor growth in the Ramos model. Interestingly, thetherapeutic effect persisted far beyond the dosing period. APX005treatment resulted in inhibition of rituximab pre-treated and resistanttumors. A dose-range finding study was performed in the Raji model andfound that minimal effective dose was determined as 1 mg/kg, and themaximal anti-tumor activity was observed at doses >3 mg/kg. In additionto B-cell lymphoma, APX005 also exhibited significant potent anti-tumoractivity in the human multiple myeloma IM-9 model.

Thus, the above Examples demonstrate that APX005 can be used to improvethe treatment of patients with NHL, CLL, multiple myeloma and certainsolid tumors that express the CD40 target. Upon binding to CD40, APX005recruits cytotoxic cells to kill tumor cells via ADCC. APX005 can alsodirectly inhibit tumor cell proliferation and activate APC via itsagonist activity. In vivo, APX005 markedly inhibited the growth ofmultiple CD40-expressing human tumor xenografts and showed long-lastinganti-tumor effects. APX005 is also capable of inhibiting human multiplemyeloma and rituximab pre-treated and resistant tumors.

Example 4 APX005 S267E Fc Mutant Increases Binding to FcγRIIB and CD40Agonist Activity

In an effort to increase binding to FCγ receptor and to increase theagonist activity of the APX005 antibody, the serine (S) at residue 267of APX005 Fc (IgG1, EU numbering) was mutated to glutamic acid (E) togenerate the APX005 S267E mutant. The mutation from AGC (S) to GAG (E)at position 267 located at APX005 heavy chain Fc region was confirmed bysequencing. The IgG1 heavy chain constant region comprising the modifiedFc region sequence is provided in SEQ ID NO:195 (this sequence includesCH1, hinge, CH2 and CH3 of IgG1).

In order to assess the ability of the APX005 S276E mutant to bind toCD40, APX005 S267E heavy chain and light chain plasmids were purifiedfrom transformed bacteria and transfected into 293-6E cell. Four daysafter transfection, antibody-containing culture supernatants werecollected and tested for binding to CD40 using ELISA (ELISA platescoated with CD40-Fc protein). The ELISA data showed that APX005 S267Emutant had similar binding activity as APX005 to CD40 (see FIG. 19).

In another experiment, the ability of the CD40 antibodies to bind tonative CD40 protein was determined by FACS analysis with live Ramoscells. Ramos cells were harvested and blocked with 1.5 ml blockingbuffer (0.1% BSA/PBS) for 30 minutes. Antibodies at variousconcentrations were added to cells and incubated for 1 hour. Afterwashing, 50 ul Goat-anti-human IgG (H+L) PE antibody at 1:1000 dilutionin blocking solution was added and incubated for 30 minutes. Cells werethen washed with PBS and were re-suspended with 1 m PBS and analyzed byFACS. The antibodies tested were the 19-21 rabbit anti-CD40 agonistantibody, SGN-40 developed by Seattle Genetics and CP870893 from Pfizer.As shown in FIG. 20, APX005 and APX005 S267E mutant showed similarbinding affinity to CD40 as SGN40, but 6 fold higher affinity thanCP870893.

An experiment was performed to compare the CD40 agonist activity ofAPX005, APX005 S267E mutant, and other anti-CD40 antibodies in a B cellactivation assay. In particular, CD40 agonist activity was tested bymeasuring the up-regulation of CD86, a B cell activation marker, onhuman B cells following treatment with various anti-CD40 antibodies invitro. Fresh human B cells were obtained by isolating B cell fromperipheral blood using a negative selection method (All Cells). B cellswere treated with different human antibodies at 10, 3.33, 1.11, 0.37,0.12, 0.04, 0.01 and 0.0045 μg/ml for 48 hours. The cells were thencollected and analyzed for human CD86 expression on live B cells by flowcytometry. 19-21, SGN-40 and CP870893 (see description above) weretested. As shown in FIG. 21, APX005 S267E mutant demonstratedsignificantly increased potency (EC50) and efficacy (EC100) as comparedto APX005. APX005 S267E was the most potent CD40 agonist antibody amongthe anti-CD40 antibodies tested in this assay.

A further experiment was conducted to assess the ADCC activity of APX005and APX005 S267E mutant. Specifically, the ability of the CD40antibodies to mediate ADCC effects in vitro was assayed using Daudicells as target cells and human PBMC as effector cells. Labeled targetcells were plated in triplicates at 5×10³ cells/well in round-bottomed96 well plates. Increasing concentration of antibodies (50 ul/well incomplete medium) was added to the labeled cells. The final antibodyconcentration ranged from 0-10 ug/ml (in complete medium). The cellswere treated with the antibodies for 30 minutes at 37° C. Effector cells(PBMC) in complete medium were seeded into target cells, (EffectorCells/Target Cell ratio=40/1). Final volume of effector cells is 100ul/well. After 4 hours incubation in a 37° C. incubator, 100 ul culturesupernatant from each well was transferred to a 96-well black view plateand read RFU with 485 ex/535 em. Specific lysis was calculated accordingto the formula: Specific lysis=[(test release-spontaneousrelease)/(maximum release−spontaneous release)]×100. Rituxan was used aspositive control. As shown in FIG. 22, the APX005 S267E mutant showedslightly increased the ADCC activity as compared to APX005 wild type.

Example 5 Characterization of the Epitope of APX005

This example describes the characterization of the epitope of APX005, ahumanized anti-CD40 antibody. As described in further detail below, atotal of 5841 linear and Chemically Linked Peptides on Scaffolds (CLIPS)peptides were synthesized and analyzed for binding. The dominant bindingregions were identified as ₉₂TSEACESCVLHRSCSP₁₀₇ (SEQ ID NO: 196) and₁₂₅PCPVGFFSNVSSAFEKCHPW₁₄₄ (SEQ ID NO: 197). Of the identified bindingresidues, T₉₂, E₉₇ and ₁₀₀VL₁₀₁ are known as being contact residuesbetween CD40 and the CD40L trimer. Specifically E₉₇ is considered animport residue for binding of CD40 to the ligand.

The CD40 protein sequence is set forth in SEQ ID NO: 198, and isdirectly available as a single crystal structure: 3QD6. Residues of CD40predicted to bind to the CD40L trimer are E74, Y82, D84, N86 and E117(see e.g., Bajorath et al., 1995 Biochemistry 34:9884-9892)) It shouldbe noted that the sequence numbering in the 3QD6 structure is 20residues higher than the numbers mentioned herein. Thus, the predictedkey binding residues are E54, Y62, D64, N66 and E97.

Synthesis of Peptides and Screening Procedures.

To reconstruct discontinuous epitopes of the target molecule a libraryof structured peptides was synthesized. This was done using Pepscan'sproprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology(Pepscan Presto, Lelystad The Netherlands) (see e.g., Timmerman et al.(2007). J. Mol. Recognit. 20:283-99; Slootstra et al. (1996). MolecularDiversity 1: 87-96). CLIPS technology provides the ability to structurepeptides into single loops, double-loops, triple loops, sheet-likefolds, helix-like folds and combinations thereof. CLIPS templates arecoupled to cysteine residues. For this experiment, the side-chains ofmultiple cysteines in the peptides were coupled to one or two CLIPStemplates. For example, a 0.5 mM solution of the T2 CLIPS template1,3-bis (bromomethyl) benzene was dissolved in ammonium bicarbonate (20mM, pH 7.9)/acetonitrile (1:1(v/v). This solution was added onto thepeptide arrays. The CLIPS template binds to side-chains of two cysteinesas present in the solid-phase bound peptides of the peptide-arrays (455wells plate with 3 ul wells). The peptide arrays were gently shaken inthe solution for 30 to 60 minutes while completely covered in solution.Finally, the peptide arrays were washed extensively with excess of H₂Oand sonicated in disrupt-buffer containing 1 percent SDS/0.1 percentbeta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followedby sonication in H₂O for another 45 minutes. The T3 CLIPS carryingpeptides were made in a similar way but with three cysteines.

The binding of antibody to each of the synthesized peptides was testedin a PEPSCAN-based ELISA. The peptide arrays were incubated with primaryantibody solution (overnight at 4° C.). After washing, the peptidearrays were incubated with a 1/1000 dilution of an antibody peroxidaseconjugate (SBA, cat.nr.2010-05) for one hour at 25° C. After washing,the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate(ABTS) and 2 microliters/milliliter of 3 percent H2O2 were added. Afterone hour, the color development was measured. The color development wasquantified with a charge coupled device (CCD)—camera and an imageprocessing system.

Data Processing

The values obtained from the CCD camera range from 0 to 3000 mAU,similar to a standard 96-well plate ELISA-reader. The results werequantified and stored into the Peplab database. The binding values wereextracted for analysis. Occasionally a well contained an air-bubbleresulting in a false-positive value. The cards were manually inspectedand any values caused by an air-bubble were scored as 0.

The determination of the epitopes was conducted through a combination of20-mer overlapping peptides and single-residue mutagenesis of 12specific areas of the protein. These 12 areas were picked based on theknown 3D structure of CD40 and on published cysteine bonds in theprotein. A total of 5841 chemically synthesized CLIPS peptides weresynthesized.

Evaluation of the overlapping 20-mer peptides indicated two distinctbinding regions: ₉₂TSEACESCVLHRSCSP₁₀₇ (SEQ ID NO: 196) and₁₂₅PCPVGFFSNVSSAFEKCHPW₁₄₄ (SEQ ID NO: 197). Evaluation of theoverlapping sequences in which two alanine-replacements were added gavesimilar results.

The 12 candidate epitope “hotspots” that were elected each contain atleast 10 identical ‘control’ peptides in which no mutation is made.Evaluation of these control sequences for each of the 12 mutagenesisdata sets identified two similar distinct binding regions:₈₄TCEEGWHCTSEACESCVLH₁₀₂ (SEQ ID NO: 199) and ₁₂₅PCPVGFFSNVSSAFEKCHPW₁₄₄(SEQ ID NO: 197). The obtained ELISA read-outs are statistically highlysignificant (p<2e⁻¹⁰). Considering the overlap of ₉₂TSEACESCVLHRSCSP₁₀₇(SEQ ID NO: 196) and ₈₄TCEEGWHCTSEACESCVLH₁₀₂, (SEQ ID NO: 199) residues92-102 (SEQ ID NO:202) are possibly the most relevant residues forbinding. Data from the CLIPS matrix set combining three differentsequence areas to identify discontinuous epitopes did not yieldadditional insights.

In-depth analysis of the 2 mutagenesis datasets(84TCEEGWHCTSEACESCVLH102 (SEQ ID NO: 199) and125PCPVGFFSNVSSAFEKCHPW144 (SEQ ID NO: 197) did not indicate anycritical residues, with the possible exception of W144. However,mutagenesis of residues 84-102 was also conducted on the linear peptide,without CLIPS (sequence 84TCEEGWHSTSEASESCVLH102 (SEQ ID NO:200), thetwo underlined residues are replaced from C to S). In this linearizedpeptide binding by APX005 almost completely disappeared, indicating thatthe binding of this antibody depends specifically on those twocysteines, or depends on the specific conformational state the peptidewas forced into by the CLIPS assembly.

Visualization of areas ₉₂TSEACESCVLHRSCSP₁₀₇ (SEQ ID NO: 196) and₁₂₅PCPVGFFSNVSSAFEKCHPW₁₄₄ (SEQ ID NO: 197) onto the crystal structureof CD40 showed that amino acids 92-107 forms a looped structure facingthe CD40L-trimer (₉₇ESC₁₀₀ is within 4 A of the ligand) (see FIG. 23).Residues 125-144 are not resolved in structure 3QD6, but based on theavailable data on cysteine bonds, are expected to be in close proximityto residues 92-107. For visualisation purposes, residues 122-125 (SEQ IDNO:201) are shown in FIG. 23.

Thus, this experiment describes the mapping of the binding of humanizedantibody APX005 onto CD40 using linear and CLIPS peptides, carried outby Pepscan Presto BV. The mapping resulted in the identification of twospecific binding regions. These are ₉₂TSEACESCVLHRSCSP₁₀₇ (SEQ IDNO:196) and ₁₂₅PCPVGFFSNVSSAFEKCHPW₁₄₄ (SEQ ID NO:197). Within region92-107, residues ₉₂TSEACESCVLH₁₀₂ (SEQ ID NO:202) are possibly the mostrelevant residues for binding. Of the identified binding residues, T₉₂,E₉₇ and ₁₀₀VL₀₁ are known as being contact residues between CD40 and theCD40L trimer. Specifically E₉₇ is considered an import residue bindingof CD40 to the ligand.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1-23. (canceled)
 24. An isolated antibody, or antigen-binding fragmentthereof, that binds to CD40, wherein the isolated antibody, or anantigen-binding fragment thereof, comprises (i) a heavy chain variableregion comprising the VHCDR1 region set forth in SEQ ID NO:3, the VHCDR2region set forth in SEQ ID NO:4, and the VHCDR3 region set forth SEQ IDNO:5; and (ii) a light chain variable region comprising the VLCDR1region set forth in SEQ ID NO:6, the VLCDR2 region set forth in SEQ IDNO:7, and the VLCDR3 region set forth in SEQ ID NO: 8; and comprising anFc region modified by a S267E mutation; or a variant of said antibody,or an antigen-binding fragment thereof, comprising heavy and light chainvariable regions identical to the heavy and light chain variable regionsof (i) and (ii) except for up to 8 amino acid substitutions in said CDRregions.
 25. The isolated antibody, or antigen-binding fragment thereof,of claim 24, wherein the antibody is humanized.
 26. The isolatedantibody, or antigen-binding fragment thereof, of claim 24, wherein theantibody is selected from the group consisting of a single chainantibody and a univalent antibody lacking a hinge region.
 27. Theisolated antibody, or antigen-binding fragment thereof, of claim 24,wherein the antibody is a whole antibody.
 28. The isolated antibody, orantigen-binding fragment thereof, of claim 24 comprising a human IgGconstant domain.
 29. The isolated antibody, or antigen-binding fragmentthereof, of claim 28, wherein the IgG constant domain comprises an IgG1CH1 domain.
 30. The isolated antibody, or antigen-binding fragmentthereof, of claim 28, wherein the IgG constant domain comprises an IgG1Fc region.
 31. The isolated antibody, or antigen-binding fragmentthereof, of claim 24, which competes with an antibody comprising the VHregion set forth in SEQ ID NO: 1 and the VL region set forth in SEQ IDNO:2 for binding to CD40.
 32. An isolated polynucleotide encoding theisolated antibody, or antigen-binding fragment thereof, according toclaim
 24. 33. An expression vector comprising the isolatedpolynucleotide of claim
 32. 34. An isolated host cell comprising thevector of claim
 33. 35. A composition comprising a physiologicallyacceptable carrier and a therapeutically effective amount of theisolated antibody, or antigen-binding fragment thereof, according toclaim
 24. 36. A method of treating or ameliorating the symptoms ofcancer in a patient, comprising administering to the patient thecomposition of claim 35, thereby treating or ameliorating the symptomsof the cancer.
 37. The method of claim 36, wherein the cancer isselected from the group consisting of non-Hodgkin's lymphomas, Hodgkin'slymphoma, chronic lymphocytic leukemias, hairy cell leukemias, acutelymphoblastic leukemias, multiple myeloma, carcinomas of the bladder,kidney ovary, cervix, breast, lung, nasopharynx, malignant melanoma andrituximab resistant NHL and leukemias.
 38. A method for amelioratingsymptoms of an autoimmune disease in a patient comprising administeringto the patient the composition of claim 35, thereby ameliorating thesymptoms of an autoimmune disease.
 39. A method for amelioratingsymptoms of inflammatory disease in a patient comprising administeringto the patient the composition of claim 35, thereby ameliorating thesymptoms of inflammatory disease.